Download - Immunoassay Buffer

There is another top address in Darmstadt:AppliChem GmbH Ottoweg 4 D - 64291 Darmstadt Phone +49 6151 9357-0 Fax +49 6151 9357-11

eMail [email protected] internet www.applichem.com

Take the Pink Link!

www. .com

Immunoassay Buffer

Take the Pink Link!

www..com

Gel Electrophoresis Size Marker

Gel Electrophoresis Size Marker

Gel Electrophoresis

Take the Pink Link!

www..com

Detergents

Take the Pink Link!

www..com

Immunoassay Buffer

Take the Pink Link!

www..com

Kontaminationen durch Nukleinsäurendurch Nukleinsäurendurch

Probleme & praktische Lösungen NukleinsäurenProbleme & praktische Lösungen Nukleinsäuren

Take the Pink Link!

www..com

WichtigesWissenswertesWunderbaresWunderbares

aus Chemie & Biologie& Biologie&

AppliCationsDer Anteil von molekularbiologischen Nachweismethoden ist in den letzten Jahren erheblich gestiegen, besonders in den Bereichen Qualitätskontrolle, Forensik, klinischer Forschung und Diagnostik – insbesondere der Infektionsdiagnostik. Gerade für diese Applikationen werden hochsensitive und gleichzeitig zuverlässige PCR-Tests benötigt. Dafür bietet AppliChem nun optimierte PCR-Kits an und widmet sich explizit der Hintergrundproblematik, die durch DNA belastete Reagenzien und Arbeitsplätze entstehen kann.

Nr.3AppliCations

Nr.3AppliCationsDNA-freie Reagenzien und Mastermixe für die PCR

AppliCationsAppliCations

Freie Nukleinsäuren verursachen als Kontaminationen große Probleme im Forschungs- und molekularbiologisch-analytischen oder klinisch-diagnostischen Labor. Durch die extrem hohe Sensitivität von DNA-Nachweistests, können kleinste Verunreinigungen in PCR-Ansätzen zusätzliche Arbeit bedeuten und im schlimm-sten Fall Ergebnisse verfälschen. Mit Derma-ExitusPlus™ (HHDK) aus der Serie von ExitusPlus™-Produkten wird erstmals ein völlig neuer Anwendungsbereich erschlossen bzw. zusätzliche Kontaminationsquellen ausgeschlossen.

Eine der Hauptquellen für Kontaminationen mit Nukleinsäuren ist der Experimentator selbst. Die Nukleinsäuren stammen z.B. aus Hautschuppen, Haaren und Speichel oder von Mikroorganismen, die seine Haut besiedeln oder z.B. beim Niesen freigesetzt werden. Gelangen diese in die PCR-Ansätze oder PCR-Reagenzien, können sie entsprechend der eingesetzten Primer (besonders 16S rDNA für Bakterien) leicht nachgewiesen werden. Ausserdem besteht die Gefahr, dass beim Öffnen und

Nr.5AppliCations

Nr.5AppliCationsDekontamination der Haut und Hände von Nukleinsäuren

AppliCations AppliCationsSize-exclusion chromatography (SEC) is a popular method to separate biomolecules based on their size. Primarily, it is applied to the separation of biopolymers such as proteins and nucleic acids, i.e. water-soluble polymers. This system is also called gel filtration, typically with beads of dextran or agarose serving as gel matrix. Smaller molecules pass significantly slower through the column than larger molecules. Not to be mixed up with gel electrophoresis, there are big differences in terms of theseparation principle. SEC does not require electric current and the sieving effect will not separate small molecules first.

It is indeed correct that smaller molecules pass more slowly through the matrix than larger molecules. This is due to the longer path the smaller molecules must travel. The longer path arises from the pores of the beads. The smaller molecules can

Keywords

No.6AppliCations

No.6AppliCationsSize-Exclusion Chromatography for purification of biomolecules

AppliCations

Take the Pink Link!

.com

Agarose-Gel-Elektrophorese AppliCations

Using ready-to-use ELISA kits from manufacturers is easy and convenient. Sometimes however, home-made ELISA is required because there is no kit available with the right antibodies or the characteristics of the available kits such as their limits of detection are not appropriate. Ready-to-use ELISA kits from good suppliers mastored for two years at 4°C without any problem. With home-made ELISA it is a completely different story. For any new measurement one has to coat a new plate, because after storage of some days the plates don’t perform as well as before.

Why is there such a great difference in storage between home-made ELISA and ELISA kits?The reason is that in professional ELISA kit production the plates are not only blocked after coating, but also stabilised. This easy to perform process has been an industry standard for thirty years. For stabilisation of a plate one has to coating stabiliser solution. It is just as simple as a “second blocking step”. But there were no such high lutions freely available in low volumes for use in research lab until now. AppliChem now offers a product for use in every researchin volumes starting as small as 50 ml, which is called the AppliCoat Plate Stabiliser (Cat. No. A7708). This stabiliser solution is easy-to-use and has a great advantage compared to almost any stabiliser used in industry. It gives better storage stability for coated antibodies and antigens than most other products do. And there is a second

Two benefits with one solutionWhen antibodies are coated onto ELISA plates, most of the antibodies are not active. When the antibodies (or any proteins) come into close contact to the plastics surface of the ELISA plate, conformational changes can occur due to surface-protein interactions. The result is that most antibodies coated on a plate are unfolded or inactive. Only around 2–8 % of all coated antibodies remain active and can bind to analytes and this is greatly variable depending on the surface characteristics of the ELISA plate, which can really differ from batch to batch or even from well to well.These differences from well to well can affect the variability of an assay, because the antibodies can be affected. If there waway of refolding antibodies and of preserving antibodies from conformational changes during storage, this could help to decrease such variabilities in assay performance. This is a key benefit of AppliCoat Plate Stabiliser. It assists antibodies and coated proteins to refold and then to preserve active conformation over a long time. Thus it has two benefits: 1. Refolding of antibody conformation of some of the coated antibodies and 2. Preserving correct conformation during storage. These benefits are used for production of high-quality ELISA kits as well as in research applications now. Even with AppliCoat Plate Stabiliser the percentage of active antibodies will still be in the range of 2–8 %. But the great difference is that the variability from well to well and from plate to plate can be minimised in most assays by using AppliCoat Plate Stabiliser. Such effects depend on the used antibodies, but when ELISA are validated (e.g. according to “Guidance for Industry: Bioanalytical Method Validation”, FDA, 2001) or according to other validation strategies, the difference can be measured in many assays.The positive effects of AppliCoat Plate Stabiliser are shown in Fig. 1. A sandwich ELISA with a monoclonal antibody has been

Keywords

Immunoassays

Antibody Stabilisation

ELISA Plates

Cross-reactivity

Interfering effects

Improving quality of ELISA

AppliCationsDie moderne Gentechnik zeigt, dass in vielen Fällen schon freie DNA-Moleküle für Infektionen, Rekombinationen oder biologische Transformationen ausreichen [1,2]. Zusätzlich werden die Nachweisverfahren für DNA-Moleküle immersensitiver. Daher wird die Detektion von Kontaminationen oder die Verhinderung von Amplifikations-Artefakten in der PCR für die Gentechnik, die Kriminalistik, die Biomedizin und die Hygiene immer wichtiger. Die vollständige Dekontamination von Geräten und Materialien von DNA-Molekülen wird so zu einem entscheidendenFaktor für die allgemeine biologische Sicherheit.

Alles oder Nichts: Erstaunliche ErkenntnisDas Mittel der Wahl zur Beseitigung von Kontaminationen durch NukleinDas Mittel der Wahl zur Beseitigung von Kontaminationen durch NukleinDas Mittel der Wahl zur Beseitigung von Kontamina säuren ist immer noch Chlorbleichlauge („bleach“) – ein Mittel das alles zerstört, nicht nur die Nukleinsäure. Dies hat uns veranlasst in Kooperation mit multiBIND Biotech, Köln, nach einer unschädlichen Alternative zu suchen und die molekulare Wirkungsweise der auf dem Markt befindlichen sonstigen DNA-Dekontaminationsmittel zu untersuchen. Hierfür wurde unter sehr hoher Belastung (großer DNA-Über-

suchen. Hierfür wurde unter sehr hoher Belastung (großer DNA-Über-suchen. Hierfür wurde unter sehr hoher Belastung (großer DNA-Überschuss) mit definierten DNA-Kontaminationen die Eigenschaften der konventionellen Mittel verglichen. Zwei Probleme werden offensichtlich: Erstens werden durch die konventionellen Mittel in keinem Fall die DNA-Moleküle effizient zerstört und zweitens enthalten diese Mittel Komponenten mit stark korrosiven oder giftigen Eigenschaften. Als Fazit daraus hat sich für uns die Notwendigkeit der Neuentwicklung einer effektiven Lösung zur DNA-Dekontamination ergeben, die wir hier als DNA-ExitusPlus™ und Autoclave-ExitusPlus™ vorstellen. Im Vergleich zu den herkömmlichen Produkten wird DNA und RNA schnell und effizient zerstört, ohne dass das Reagenz korrosive oder giftige Eigenschaften aufweist.Bei der DNA-Dekontamination unterscheidet man nach der molekularen Wirkungsweise der eingesetzten Mittel drei Grund-prinzipien zur Zerstörung oder Inaktivierung der genetischen Information: Modifikation, Denaturierung und Degradation.

prinzipien zur Zerstörung oder Inaktivierung der genetischen Information: Modifikation, Denaturierung und Degradation.

prinzipien zur Zerstörung oder Inaktivierung der genetischen Information: ModifikaJe nach Zusammensetzung der Mittel können diese drei Prinzipien einzeln oder in Kombination angewandt werden.Da nach den aktuellen Erkenntnissen zum biologischen Risikopotenzial von freien DNA-Molekülen für eine wirklich sichere DNA-Dekontamination die Zerlegung dieser DNA-Moleküle in möglichst kleine Fragmente die wirkungsvollste Methode ist, wurden die gängigen konventionellen Mittel mit unserer Neuentwicklung DNA-ExitusPlus™ im DNA-Degradationstest ver-tionellen Mittel mit unserer Neuentwicklung DNA-ExitusPlus™ im DNA-Degradationstest ver-tionellen Mittel mit unserer Neuentwicklung DNA-ExitusPlus™ im DNA-Degradationstest verglichen. Der DNA-Degradationstest erlaubt einen sensitiven, quantitativen Vergleich der Geschwindigkeit des DNA-Abbaus (Abb. 1 und 2).

Unerwarteter Weise haben wir festgestellt, dass einige der bekannten kommerziellen Mittel nur mit dem Prinzip der Modifi-kation oder Denaturierung der DNA-Moleküle arbeiten. Eine Zerlegung der DNA-Stränge erfolgt dabei nicht, sondern die genetische Information, für die diese DNA-Stränge kodieren, wird eigentlich nur maskiert. Eine genetische Information, für die diese DNA-Stränge kodieren, wird eigentlich nur maskiert. Eine genetische Informa

chemische Demaskierung der DNA-Moleküle durch Entfernung der blockierenden Gruppen würde die genetische Information wieder lesbar und am-plifizierbar machen. Nach dem heutigen Wissensstand zur Gentechnik und der Problematik der Neukombination von Erb-trägern sind solche Mittel eigentlich nicht mehr zeitgemäß. Aber auch die Mittel, die zu einer nachweisbaren Degradation

Keywords

Nukleinsäure-Dekontamination

DNA-DegradationstestPCR-Test

Autoklavieren von DNA

Nr.1AppliCations

Nr.1AppliCationsNukleinsäure-Dekontamination mit der ExitusPlus™-Technologie

AppliCations

Take the Pink Link!

www..com

TransferMembranes

Take the pink Link!

www..com

Take the pink Link!

www..com

Ever since the foundation of the firm, AppliChem has

invested extensively in communication and marketing. The

fresh and unusual appearance attracted great attention in

the market from the very beginning. Our growth confirms

the relevance of these measures.

We offer our customers an extensive library, with numerous

brochures and applications whose use makes everyday life

in the laboratory easier.

A comprehensive catalogue of products, with detailed

information provided by no other catalogue in the field,

is available in German and English.

AppliChem brings advantages through knowledge.

“Thinking without knowledgemakes chance the ruler.”Werner Kollath, German bacteriologist

Limiting Cross Reactivity in Immunoassays 2

Without Blocking .... No Result! 10

Tips for storing antibodies 12

Comparing Blocking Reagents 14

Improving quality & stability of ELISA 18

Products with application notes

Antibody Stabilizer-PBS 21

Antibody Stabilizer-Tris 21

Applicoat Plate Stabilizer 22

Blocking Buffer I 23

Blocking Buffer II EGrade 24

Blocking Buffer III BSA 25

Blocking Reagent CA 26

Coating Buffer pH 9,6 27

Coating Buffer pH 7,4 27

CrossDown Buffer 28

CrossDown Peroxidase-Stabilizer 31

Peroxidase-Stabilizer 32

Sample Buffer T- 33

Sample Buffer T+ 33

Stripping Buffer I 34

Washing Buffer TrisT- (10X) 34

Washing Buffer TrisT+ (10X) 35

Related products 36

Further reading 36

© 2010 AppliChem • Immunoassay Buffer 1

contents

2 Immunoassay Buffer • AppliChem © 2010

immunoassay

Immunoassays are a very important tool in bioanalytical and biochemical laboratories. They are used in research, food and environmental monitoring as well as in diagnostic applications. Immunoassays are quite easy to carry out and very specific in terms of quantitative and qualitative significance due to their use of antibodies for detection. Theoretically, each antibody can identify one antigen and binds this antigen with high affinity which explains why one can distinguish so easily between different substances.

In practice, it is not that simple. Immunoassays suffer from cross reactivity which results in false bands in Western blots, signals in the negative control of an ELISA or a very high background in a protein array. Every false result means more work, additional costs and potentially misdiagnosis of patients [1]. Although antibodies are very specific and have high affinity for one antigen in particular, often antibodies can also bind with lower affinity to other antigens which are not detected by the assay. This is even observed with very

Fig. 2A Non-specificbindingofalabeleddetectionantibodytoanot(sufficiently)blockedsurface.Result:false-positivesignal.

B Non-specificbindingofalabeleddetectionantibodytoablockedsurface.Despiteblockingofthesurfacetheantibodybindstotheblo-ckingproteinitself.Result:false-positivesignal.

C AninterferingproteinbindstotheFcsegmentofthedetectionantibodyandhindersstericallythebindingoftheanalyte.Result:false-negativesignal.

D ThecaptureantibodybindstotheFcsegmentofthelabeleddetectionantibody.Theanalytecan-notbeboundbythecaptureantibodyanymore.Result:false-positivesignals

Fig. 1Theperfection:interference-freesandwichassay.

Dr.WolframH.Marx,AppliChem;PDDr.Wiesmann,UniversityofMünster;Dipl.Biol.SusanneSiewert,UniversityofUlm;Dipl.Chem.NicoDankbar,UniversityofMünster;Dr.PeterRauch,CANDORBiosciennceGmbH;Dr.ChristophSpechtPARA,BioscienceGmbH

labeled detection antibody

analyte

capture antibody

interfering or cross reacting substance

heterophilic antibodies or HAMA

blocking protein of surface

immunoassayLimiting Cross Reactivity in Immunoassays

There are several types of these assays including Enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), Western blotting, radioactive labelled immunoassays (RIA), protein arrays, immunohistochemistry, and immunopolymerasechain reaction (ImmunoPCR). Each of these assays have one drawback in common cross reactivity.

Antibodies are used in Immunoassays to easily and specifically distinguish between different substances.


well characterised antibodies known to have a high affinity to the target analyte.

The result are interferences such as nonspecific binding, cross reactivities and matrix effects leading to high background with bad signaltonoise ratio.

Interferences in immunoassaysInterference can come in several forms such as cross reactivities, nonspecific binding and matrix effects. Laboratory or clinical samples may contain foreign substances in concentrations that can interact with the analyte or the capture/detection antibodies thereby disrupting the desired reaction. Similarly, surfaces that act as platforms for immunoassays have also been known to be a source of interference. By applying novel buffers (e.g. CrossDown Buffer), most of the above mentioned effects can be avoided. Simply exchange the sample buffer or antibody dilution buffer by CrossDown Buffer and thereby improve the quality of the assays and the efficiency of the assay development.

To understand the basic principles of interference, we first take a look at an ideal sandwich ELISA (fig. 1) followed by fig. 2–5 where interference is demonstrated. In a troublefree sandwich ELISA, the capture antibody is immobilised on the bottom of a well. The rest of the surface is blocked sufficiently. The capture antibody binds the analyte, while the secondary labelled antibody, binds to a different site on the analyte (fig. 1).

Non-specific bindingNonspecific binding occurs when an antibody binds to substances present in much higher concentrations than the target analyte (e.g. nonspecific binding to albumin or immunoglobulins), binding to surfaces (e.g. Western blotting membranes or ELISA wells), or binding to loci on immobilized antibodies in protein arrays [2]. Assays with insufficient blocking or with difficult matrices containing e.g. a high albumin concentration or high concentration of endogenous interfering substances, are strongly affected. There are also other causes of nonspecific binding.

Since detection antibodies are labelled with enzymes (e.g. alkaline Phosphatase or Peroxidase), fluorescent dyes, radioactive isotopes or DNA (ImmunoPCR), the label itself can also be a source of unwanted interactions.

In the case of fluorescent dyes, which are frequently hydrophobic, the binding properties of detection antibodies can be changed. The dyes themselves may cause unwanted binding and thereby reduce the solubility of the labelled protein. The antigenantibody binding can be impaired too [3]. These effects may lead to increased nonspecific binding of the labelled antibody onto surfaces (fig. 2A and 2B), to foreign proteins of the real sample (fig. 2C) or to the capture antibody (fig. 2D). In those cases false positive results are obtained even in the absence of an analyte or the whole assay shows a high background, respectively. In protein arrays this phenomenon leads to higher background fluorescence of single spots or a low signaltonoise

Fig. 3I Crossreactivityofaninterferingsubstancewiththecaptureantibody.Result:false-negativesignal.

J Crossreactivityofaninterferingsubstancewiththedetectionantibody.Result:false-negativesignal.

K Crossreactivitybothwiththecaptureandwiththedetectionantibody.Result:false-positivesignalsSuchaphenomenonisratherseldominpractice,butdefinitelypossiblewithantibodieshavinglowerspecificity.Suchaninterferencepicturemayoccurwithantibodiesdirectedtoatargetwithaconservedaminoacidsequenceofaproteinwhosesequencemotivealsooccursinotherproteins.

Fig. 4F BridgingbyHAMAsandheterophilicantibodies,respectively,resultinginacouplingofthecaptureanddetectionantibody.Result:false-positivesignals.

G Ananti-idiotypicHAMAbindingtothecaptureantibody.TheinterferingantibodybindsintheareaofthehighlyvariableregionoftheFabsegmentandthuspreventsthebindingoftheanalyte.Result:false-negativesignal.

H Ananti-idiotypicHAMAbindingtothedetectionantibody.TheinterferingantibodybindsintheareaofthehighlyvariableregionoftheFabsegmentandpreventsthebindingoftheanalyte.Result:false-negativesignal.

Fig. 5L Maskingoftheanalytebyaproteinofthespecimen.Theepitopeisblockedforbindingofthecaptureantibody,resultinginnobindingtotheanalyteatallorinthecaseofastericalhindrancebindingisveryweek.Result:false-negativesignal.


immunoassayratio, respectively. Fluorescent dyes also may bind proteins or antibodies from serum samples, resulting in a reduction of the dye fluorescence and in extreme cases to a complete quenching of the signal. Based on this, discussions are ongoing to eliminate fluorescent dyes from protein arrays completely [4]. The complexity of protein arrays is very high due to the application of many different capture antibodies and labelled detection antibodies in one reaction. Thereby, the risk of nonspecific binding of proteins in the sample or labelled antibodies to single spots increases significantly as well as interferences of components of the sample with the antibodies [2].

Cross reactivityCross reactivities are the result of nontarget binding and appears similar to nonspecific binding. Unlike nonspecific binding, however, when one talks about cross reactivity the cross reacting substance is known and its cross reacting properties can be proven e.g. by measurement of the competing concentration of the cross reacting species [5]. Cross reactivity is the ability of the antibody to bind structures other than the target analyte (fig. 3I, 3J and 3K). Often, these structures are similar to the analyte such as metabolites or chemical substances with a similar molecular structure. Proteins with evolutional homology of amino acid sequence or similarity in tertiary structure can cross react too. Cross reactivities play a key role in many competitive assays, because only one antibody is used [1, 4]. For these assays, it is part of the validation to identify and to quantify possible cross reactors experimentally [5].

Cross reactivities can also play a major role in the detection of proteins in Western blots or in immunohistochemical applications. Cross reactivity can result in staining of additional bands in Westerns or cell structures, without knowing the exact molecular reasons for this unwanted binding. In Western blots, sometimes the additional bands simply represent protein fragments which originate from the “normal” degradation process. But in some cases it is important to look closer at cross reactivities caused by the primary or secondary antibody. For any scientific publication it is necessary to verify the reasons for unexpected bands and signals anyway.

Matrix effects

The least defined term is the “matrix effect”. Matrix effects are the sum of all negative effects of all components within a sample, which can affect the determination of the target analyte [6]. If the exact molecular cause of such an effect is unknown, but can be related to the composition of the sample to be determined, one

speaks about a matrix effect. There is a smooth transition to all other negative effects. Matrix effects can be caused by AntiAnimalAntibodies, heterophilic antibodies, endogenous interfering substances or influences like viscosity, pH or salt concentration.

There are negative effects that are restricted to medical and diagnostics assays. These effects are based on interfering substances present in human specimen like plasma, serum or tissue samples. Since the results of these assays are the basis of patient therapies, interferences and false results are severe.

Anti-Animal-AntibodiesHuman AntiAnimalAntibodies (HAAA) are of the IgG, IgA, IgM or IgE type and are formed as an immune response after contact with animal immunglobulins. HAAAs are well known from diagnostic assays. Studies report that up to 80 % of all samples contain HAAAs. The concentrations can be very high, reaching levels of several milligrams per milliliter [7].

HumanAntiMouse Antibodies (HAMA) are the best known interfering antibodies in immunoassays. HAMAs are human antibodies which are relatively specific and which can bind mouse antibodies with a middle affinity up to, in some rare cases, a high affinity. One of the reasons for the development of these antibodies can be nonhuman therapeutic antibodies which are administered as drugs (e.g. in cancer therapies). After therapeutic medication the human immune system reacts to these foreign antibodies and begins to produce antibodies against these mouse antibodies. Therefore, HAMAs interfere with immunological methods which include mouse antibodies. In sandwich ELISAs based on monoclonal mouse antibodies, capture and detection antibody will be bridged (fig. 4F), resulting in a false


positive signal. Due to the sequence homology between antibodies of different species, HAMAcontaining sera may disturb assays which contain antibodies from other species.

Drugs aren't the only reason for the development of HAMAs. Contact with domestic animals over several years enhance the formation of anti animal antibodies that either bind to antibodies of a single species (e.g. rabbit, mouse, dog, hamster) or binding to antibodies of several species with different affinities. Some interfering antibodies can bind to the Fc portion of the antibody, while others bind to the Fabfragment resulting in reduced binding of the analyte or even completely preventing the formation of any real complexes. The consequence is a false negative measurement (fig. 4G and 4H). The ability of HAAAs to bind the Fcfragment is called antiisotypical interference. In contrast, antiidiotypical interfering antibodies bind the highly variable, Fab portion of the antibody [7].

Heterophilic antibodiesAccording to Taber’s Medical Dictionary, 'heterophilic antibodies are antibodies which bind other antigens than the specific antigen'. Heterophilic antibodies can be of the IgG, IgM, IgA or IgE type. The IgM type plays a key role in sera from rheumatic patients. These sera do contain so called rheumatic factors in high concentration. Rheumatic factors are IgM type antibodies which bind to Fcfragments of human antibodies, and therefore they may bind to the Fcfragments of antibodies of other species in the assays as well. Rheumatic sera lead to a linkage of capture and detection antibodies with the consequence of falsepositive signals. This reflects the general interfering mechanism of heterophilic antibodies. The effect of the rheumatic sera is similar to the effect of HAAAs. The difference between HAAAs and heterophilic antibodies is their formation. The latter aren't formed upon contact with animal immun globulins, but rather they are multispecific antibodies of the early immune response or interfering antibodies with unknown immunological origin [7].

Interference by HAAAs or by heterophilic antibodies have been known now for more than 30 years. In general, the interfering antibodies are weakly binding antibodies [7], which predominantly disturb assays that, due to the low concentration of the analytes, require a low dilution of serum or plasma specimens [8]. Addition of blocking substances to the sample buffer, e.g. nonspecific sera, antibody fragments or high concentrations of animal immunoglobulins, are able to reduce the negative effects of the HAAAs or heterophilic antibodies by competition, but don’t always prevent them [7].

Interference caused by endogenous components of the specimenEven naturally occurring proteins found in specimens can interfere with immunoassays. Some well known interfering substances in human sera are albumins, complement factors, lysozymes and fibrinogen [4]. Since analytes of low molecular weight can bind readily to albumin, this reduces the accessibility of the antibody to the analyte. Numerous hormones are bound to transport proteins, which may lead to difficulties as well. The binding ability of certain proteins is a substantial part of their biological function, e.g. albumin, complement and Creactive protein (CRP). Because these proteins are natural receptors for many substances, nonspecific binding or even cross reactivity is possible which complicates the recognition of certain analytes in an assay similar to antibodies. Endogenous proteins can bind as interfering factors to antibodies (fig. 2C, 3IK) or mask the target analyte (fig. 5L). For example, lysozyme binds nonspecifically to any proteins with a low isoelectric point. Therefore, antibodies which have


immunoassay staining of the cultures intensifyes with time (fig. 6 lower left).

In many cases the substitution or optimisation of the blocking reagent alone is not sufficient, because the blocking agent has limited influence on all the different negative effects. The solution to combating the one common feature of many interfering substances, i.e. low to medium affinity binding, was used to develop a new buffer.

CrossDown Buffer (AppliChem) capitalizes on the fact that the binding of interfering substances is weaker than the specific binding of the target analytes. It eliminates low and medium affinity binding, without negatively affecting high affinity binding and high specificity. Figures 7 to 10 show different examples of typical interference effects in immunoassays that are prevented by the use of CrossDown Buffer.

Figure 7 shows a Western blot with high background. One of the typical daily problems in many labs. Only the substitution of the blocking reagent and additionally substitution of the antibody dilution buffer led to an analyzable result: Myostatin (GDF8; 12 kDa) from mouse myoblasts (C2C12) was blotted on nitrocellulose NC45 (Serva) and detected with antiGDF8 (Santa Cruz). Originally, blocking was performed with 2 % nonfat dried milk powder and 1 % BSA in TBS. As antibody dilution buffer 0.3 % BSA in TBS was applied and detection was done with ECL (Amersham). Applying the conventional protocol, the bands are hardly visible. With the substitution of Blo cking Buffer I and CrossDown Buffer as new antibody dilution buffer, a significant reduction of background is achieved (fig. 7).

Positive results in a protein chip application are shown in fig. 8. CrossDown Buffer reduced a high background and improved the signaltonoise ratio from 3.4 to 17.3. In this experiment different polyclonal anti EPIL antibodies (EPIL – early placenta insulin like growth factors) were tested for their suitability. The purified antibodies were immobilized on aminosilanefunctionalized microarray slides using a spotter (GMS 417) at a concentration of 500 µg/ml in a volume of 1.8 nl/spot. Afterwards, 2 ml supernatant of an EPILoverexpressing cell line (SKBR3) were mixed with the dye Oyster650P (Denovo Biolabels) and all proteins of the mixture were labeled. The incubation on the slide was carried out at a dilution of the medium : buffer 1 : 20 with CrossDown Buffer in comparison to PBS. After washing of the slides they were analyzed with a fluorescence scanner (GMS 418) and the data were evaluated with ImaGene (Biodiscovery Inc.). The use of CrossDown Buffer resulted in a clear reduction of the background signal, allowing the selection of antibodies in terms of their suitability to detect EPIL.

An example of the impact of a matrix effect on an ELISA is shown in figure 9. With this model assay a

an isoelectric point of approximately 5, can be bound and form a bridge between capture and detection antibody [4].

One other important aspect, which should be mentioned, is the interference by strongly fatty specimens, because some analytes are fatsoluble and the binding between antibody and analyte can be affected by lipids.

Avoiding the interference by applying novel immunoassay buffers – examples from the practiceIn most cases, the problems in many immunoassays are caused by low to medium affinity bindings. The best known strategy to circumvent the negative effects is an optimised blocking procedure. To get the systems running many blocking solution were developed. Most of them can be called 'very creative', but rather lack real good results in practice. The larger the analyte, the easier is the blocking. Small analytes often require a more efficient blocking. The optimal blocking buffer shall be a generally applicable solution for most immunoassays and shall give reliable results. Only such a multipurpose solution can help saving time and money for optimizing and developing immunoassays. This holds true especially if expensive antibodies or difficulttoprepare samples are applied. Caseinbased blocking solutions have proven to be very efficient. But preparing such a solution with consistent blocking efficiency requires a great deal of time and experience. The reason is simple. Simply solubilizing casein doesn’t give a good blocking solution. That kind of casein blocker is available from many suppliers, but the results in assays are not of the same quality like they should be. Literature describes and practice shows that casein works best, if it is cut into fragments of different molecular weights. Nowadays, a chemical modification during the manufacturing process allows the production of casein solutions with reproducible and reliable results.

Replacement of an unsuitable blocking reagent in immunohistochemistry makes the interpretation of an experiment with osteoblast culture possible (fig. 6). On the first day, a freshly prepared osteoblast culture shows no or a very weak expression of the extracellular matrix protein osteocalcin. By applying a novel caseinbased blocking reagent (Blocking Buffer I, AppliChem) in combination with antiosteocalcin (monoclonal, TaKaRa), the actual expression is correctly detected (fig. 6 upper left). Standard blocking with BSA leads to a completely falsepositive result (fig. 6 upper right). With time, the cultured osteoblasts build up the extracellular matrix and osteocalcin is synthesized. The expected increase in osteocalcin expression can be correctly monitored by using Blocking Buffer I. The


Fig. 6 Osteoblastculturefordetectionofosteocalcin.WhereasblockingwithBSAgivesawrongresult(upperright),switchingtoanovelcasein-basedblocker(BlockingBufferI,AppliChem)showscorrectresults(upperleft).Thetimecourseofexpressioncanbeshowncorrectly(lowerpanels).(imagesbyPDDr.Wiesmann,UniversityofMünster,Germany)

Fig. 7Westernblot.LeftsidewithoutandrightsidewithBlockingBufferIandCrossDownBuffer.Detectionofmyostatininmousemyoblastswithanti-GDF-8asprimaryandrabbitanti-goatIgG-HRPassecondaryanti-bodyonanitrocellulosemembraneNC45.(Dipl.Biol.S.Siewert,UniversityofUlm,Germany)

with Standard Assay Buffer with CrossDown Buffer

Fig. 8Reductionofanon-specificinteractionofthedetectionantibodywiththearraysurfacebytheuseofCrossDownBuffer.Signal-to-noisecouldbeincreasedfrom3.4to17.3.(Dipl.Chem.N.Dankbar,UniversityofMünster,Germany)


immunoassaymatrix effect was induced systematically. The assay was performed by Candor Bioscience GmbH, a company, which develops and validates assays for pharmaceutical research and diagnostic applications. Rabbit serum was used as a matrix and spiked in defined concentrations with human Creactive protein (CRP, Biotrend). As capture antibody, Clone C2 was used (Biotrend, 1 µg/ml coating concentration in PBS) and for detection, the biotinylated antibody from Clone C6 (Biotrend, working concentration 2 µg/ml) was applied. The spiked serum samples were diluted either with a PBSBSA buffer or with CrossDown Buffer 1 : 2 and measured by ELISA. Detection was carried out with NeutrAvidin™ – conjugated horseradish peroxidase (Pierce, working concentration 0.05 µg/ml in PBSBSA buffer) with ImmunoPure®TMBsubstrate (Pierce).

A matrix effect, whose exact molecular reason is not known, leads to a calibration curve with low sensitivity. Due to its physiological function, CRP is able to bind many proteins and substances (scavenger function of CRP), probably causing a significant reduction in the accessability of the epitope by the antibody. Presumably, an interfering effect as shown in figure 5L takes

place, although interfering effects as shown in figure 3IK can not be excluded. Again, CrossDown Buffer prevented the binding of CRP to endogenous substances of the rabbit serum and thus improved the sensitivity of the calibration curve by the factor of 3 (fig. 9).

The ELISA shown in fig. 10 is an example, where the substitution of sample and antibody dilution buffer by CrossDown Buffer was sufficient to achieve a good result. As antigen a lysate of human kidney carcinoma cells were immobilized and a serial dilution of two immun sera in repeat determination (1:50 to 1:36450) A–G loaded in columns 1–4. The corresponding preimmun sera (1 : 50) were pipetted in lane H. Blank values are in column 5. The result of using the standard buffer (PBS/NaCl/Tween® 20) in contrast to the new CrossDown Buffer is selfevident. It leads to a better sensitivity by reducing the level of detection (LOD) from 0.051 to 0.022 and the level of quantification from 0.152 to 0.065 and enlarging the measurement range. The improvement can be explained by the elimination of the falsepositive signals of the preimmun sera and the reduction of the background.

Fig. 9ELISAofCRPinrabbitserum(developedbyP.Rauch,CandorBioscienceGmbH).CrossDownBufferimprovedsensitivitybyavoidingamatrixeffect.

Extin

ctio

n 45

0 nm

•PBS/BSA-Standard buffer CrossDown Buffer

CRP [ng/ml]


1 2 3 4 5 1 2 3 4 5

Abb. 10 ELISAwithlysatesofahumankidney-cellcarcinoma.LeftsidePBS/NaCl/Tween®20andrightsidewithnovelCrossDownBuffer(AppliChem)LaneA-Gserialdilutionofimmunsera;LaneH:preimmunsera;Column5:blank.(Dr.Specht,ParaBioscienceGmbH).

ConclusionThe phenomenon of interference in immunoassays is as old as the application of antibodies for bioanalytical and diagnostic purposes. During the last 30 years numerous molecular causes were found and the mechanism of interference investigated which led to the development of prevention strategies. At today’s state of the technology, many interference effects can be minimized and innovative buffers for immunoassays make an essential contribution to it. In fact one can say, that “good solutions” for these problems have been developed. It is new that the same sample and antibody dilution buffer allows to minimize different interference effects with different molecular principles at the same time. CrossDown Buffer is applicable for different immunoassays.

The results shown here cover just a part of the different negative effects in different methods, which could be minimized or even avoided with this novel buffer. In addition, nonspecific binding in immunohistochemical applications and falsepositive binding in immunoPCR can be prevented. The novel Blocking

Solution I, which is manufactured in reproducible quality with its wide spectrum of fragments of different molecular weights, can help to increase the efficiency of these methods. Taken together, costs and time for optimizing assays can be avoided and reduced, respectively, as well as reliability improved.

Literatur [1] Miller, J.J. (2004) Clinical Laboratory International 28,

(2), 14-17

[2] Kusnezow, W., Hoheisel, J.D. (2003) J. Mol. Recognit. 16,

165-176

[3] Patton, W.F. (2000) Electrophoresis 21, 1123-1144

[4] MacBeath, G. (2002) Nat. Genet. 32, 526-532

[5] Miller, J.J., Valdes, R.Jr. (1992) J Clin Immunoassays 15,

97-107

[6] Wood, W.G. (1991) Scand. J. Clin. Lab. Invest. Suppl. 205,

105-112

[7] Kricka, L.J. (1999) Clinical Chemistry 45,(7), 942-956

[8] Span, P.N., Grebenchtchikov N., Geurts-Moespot, J., Sweep,

C.G.J. (2003) Clinical Chemistry 49,(10), 1708-1709


blockingWithout Blocking ... No Result! This is a short message describing the necessity to block surfaces in immunoassays.

All kinds of immunoassays require blocking to prevent nonspecific binding of antibodies or components of the sample to surfaces e.g. ELISA plates or Western blot membranes. Otherwise, this binding would lead to a strong background, falsify or even destroy results. Efficient blocking means no areas on surfaces are available for nonspecific binding. That's the theory. In practice, the devil is in the details. A review of the literature shows that for every detection method using anti bodies, several hundred blocking protocols exist describing variations of blocking solutions based on different blocking reagents. They all have in common that certain molecules are present in vast excess to cover the entire surface.

Frequently used blocking reagents contain BSA, gelatin from fish, nonfat dried milk, casein or synthetic molecules. Unfortunately, the optimum reagent has to be determined for each new assay, since they all have certain restrictions when used with real samples such as blood, serum, cell lysates or tissue sections.

Ideal BlockingWhat properties are a must for the ideal blocking reagent? First, it has to completely cover the surface and this is achieved best, if it contains molecules of dif ferent sizes where large and small gaps will be covered simultaneously. Secondly, the blocking reagent shall not react with or bind to any components of the sample or the antibodies. BSA blocking reagents

pose special problems since many analytes are coupled to BSA to achieve a better immunization. It is no wonder that some antibodies will bind with high affinity to BSA. In this case, blocking would lead to an even increased background due to the reaction of antibodies with the blocking reagent.

Highly purified Caseinbased reagents have proven to meet most of the important criteria of the ideal blocker but the preparation of such a reagent requires experience of a skilled person. Hydrolysis of casein must be performed over many hours and any mishandling may lead to precipitation of casein.

Blocking Buffer I - ready-to-useAppliChem now offers Blocking Buffer I which meets all criteria. It is based on highly purified, chemically modified casein with an optimum distribution in terms of fragment size. This ready-to-use buffered solution requires no assay optimization and is stabilized with ProClin® 300 instead of toxic additives like thimerosal or sodium azide.

Nevertheless, even effective blocking may not prevent some background, caused by matrix effects or cross reactivity. Additionally applying AppliChem's CrossDown™ Buffer shall reduce or even abolish negative effects in different immunoassays such as immunohistochemical staining, Western blots or ELISAs.

Dr.WolframH.Marx,AppliChem,Dr.AstridVoigt,UniversityHospitalJena,Dr.TronhungQuang,Dr.RainerKlocke,Prof.Dr.SigridNikol,UniversityHospitalMünster,Dr.ChristophSpecht,PARABioscienceGmbH

Many different blocking protocols exist and they work in various assays – sometimes better, sometimes worse. However, universal blocking solutions do not exist.


ImmunohistochemistryTheantigenNestinwasdetectedbyABC-immunocytochemicalstainingwithalkalinephosphataseonCytospinprepsoftheneuroblastomacelllineSK-N-LO.Whileastandardblockingbufferbasedon1%BSAinPBSstainedlargepartsofthewholesurface(upperpanel),usingtheready-to-useBlockingBufferIledtoareductionofbackgroundstaining(lowerpanel).CorrectstainingofthecytoplasmicNestinisnowclearlyseparatedfromthehematoxylinstainingofthenucleus.

Western-BlotDetectionoftheantigenCaMKIIinlysatesofheartandbraintissueofC57Bl/6micebyWesternblotting.AfterseparationbySDS-PAGE,proteinsweretransferredtoanitrocelluloseOptitranBA-S83mem-brane(Schleicher&Schuell)anddetectedbyECL.Theprimaryanti-bodywasamonoclonalanti-CaMKIIantibody(BDBioscience),andthesecondaryantibodyapolyclonalHRP-conjugatedantibody(SantaCruz).Blockingwith5%non-fatdriedmilkinTBS-Tween®isshownontheleftside.ThisWesternblotcannotbeinterpreted.AcombinationofBlockingBufferIandCrossDown™Bufferasantibodydilutionbufferfortheprimaryandsecondrayantibody(rightside),madetheidentificationofthebandspossible.

Just theory? This is the practice!

ELISAThisELISA(developedbyPARABioscience,Gronau,Germany)detectsimmunoglobulinsfromguineapigandisusedforimmunotoxicologicalstudieswithguineapigs.Thefalse-positivebindingobservedinthecontrollaneA1–A12andtheblindvalues(lane H1–H12)preventsinterpretationoftheassay.

with Standard Assay Buffer

UseofCrossDown™Bufferabolishesfalse-positivebindingandallowsaconcentration-dependentdetection(lanesB to G1–6andB to G7–12).ThecaptureantibodyGoat-anti-guineapig-IgGF(ab')2anddetectionantibodyGoat-anti-guineapig-IgGF(cγ)biotinylatedwerefromJacksonImmunoResearchLaboratories,Inc.(concentrationrangeeach0.31–10µg/mlinPBS).GuineapigIgGwasdilutedeitherinCross-Down™BufferorPBS(columns1–650ng/ml;columns7–1210ng/ml).PBS-BSAbufferwasusedasablockingbufferanddetectionwascarriedoutwithstreptavidin-peroxidase(Sigma)andortho-phenylene-diamin(Sigma).

with CrossDown

false-positive binding

false-positive binding

heart brain heart brain


12 Immunoassay Buffer • AppliChem © 200812 Immunoassay Buffer • AppliChem © 2008

Selection Guide ELISA, EIA, RIA Western Blot Protein Array Immunohisto-chemistry Immuno-PCR

CrossDown Buffer

Sample Buffer T+

Sample Buffer T-

Blocking Buffer I

Blocking Buffer II EGrade

Blocking Buffer III BSA

Washing Buffer TrisT+

Washing Buffer TrisT-

Coating Buffer

Stripping Buffer I

tips & tricks Which substances may be added to antibody-containing solutions as preservatives?Frequently, in biomedical laboratories sodium azide (final concen-tration 0.02-0.2 %) or Thimerosal (final concentration 0.005 %) are added to reagents. Both substances are toxic and less harmful alternatives are wanted. For such applications, ProClin® 300 may serve as a substitute, if growth of bacteria and fungi/molds has to be prevented.

Does it make sense to add albumin?Principally, it is good to add albumin, because albumin stabilizes antibodies. For long-term storage of antibodies in solution, we recommend the use of our Antibody Stabilizer (prod. no. A7148 and A7135), because they contain special ingredients stabilizing the structure.

Is it possible to store antibodies in 50 % glycerol?

If you really want to freeze antibodies, we recommend testing the performance of the antibody after thawing, because nearly every antibody cannot be frozen without losing part of its activity. In case the test is positive, prepare small aliquots and freeze. Don’t re-freeze and thaw again, as the loss of acitivity will be greater with every freeze and thaw cycle. Be careful during thawing, as thawing too fast may damage your antibody in solution.

A modern way of long-term storage of antibodies is storage in our reagents Antibody Stabilizer-PBS or -Tris. You may dilute the antibody in Antibody Stabilizer and directly store at 2-8°C. Most antibodies show extremely long shelf lives, if stored in Antibody Stabilizer of around 5 years or more. One of the main advantages is that you don’t have to aliquot for storage. For any new assay, you simply may take just the quantity of antibody you really need right now. The antibody is always ready-to-use for your next experiment as you don’t have to slowly thaw.


Customer No. (if known)

Title | First Name | Name

Department

Institute

University | Company

Address

ZIP Code City | State

Country

Phone Fax

eMail

Give it to me!AppliCations

Using ready-to-use ELISA kits from manufacturers is easy and convenient. Some-times however, home-made ELISA is required because there is no kit available with the right antibodies or the characteristics of the available kits such as their limits of detection are not appropriate. Ready-to-use ELISA kits from good suppliers may be stored for two years at 4°C without any problem. With home-made ELISA it is a completely different story. For any new measurement one has to coat a new plate, because after storage of some days the plates don’t perform as well as before.

Why is there such a great difference in storage between home-made ELISA and ELISA kits?The reason is that in professional ELISA kit production the plates are not only blocked after coating, but also stabilised. This easy to perform process has been an industry standard for thirty years. For stabilisation of a plate one has to incubate with a coating stabiliser solution. It is just as simple as a “second blocking step”. But there were no such high quality stabiliser so-lutions freely available in low volumes for use in research lab until now. AppliChem now offers a product for use in every research lab in volumes starting as small as 50 ml, which is called the AppliCoat Plate Stabiliser (Cat. No. A7708). This stabiliser solution is easy-to-use and has a great advantage compared to almost any stabiliser used in industry. It gives better storage stability for coated anti bodies and antigens than most other products do. And there is a second benefit with this product:Two benefits with one solutionWhen antibodies are coated onto ELISA plates, most of the antibodies are not active. When the antibodies (or any proteins) come into close contact to the plastics surface of the ELISA plate, conformational changes can occur due to surface-protein interactions. The result is that most antibodies coated on a plate are unfolded or in active. Only around 2–8 % of all coated antibodies remain active and can bind to analytes and this is greatly variable depending on the surface characteristics of the ELISA plate, which can really differ from batch to batch or even from well to well.These differences from well to well can affect the variability of an assay, because the antibodies can be affected. If there was a way of refolding antibodies and of preserving antibodies from conformational changes during storage, this could help to decrease such variabilities in assay performance. This is a key benefit of AppliCoat Plate Stabiliser. It assists antibodies and coated proteins to refold and then to preserve active conformation over a long time. Thus it has two benefits: 1. Refolding of antibody conformation of some of the coated antibodies and 2. Preserving correct conformation during storage. These bene-fits are used for production of high-quality ELISA kits as well as in research applications now. Even with AppliCoat Plate Stabiliser the percentage of active antibodies will still be in the range of 2–8 %. But the great difference is that the variability from well to well and from plate to plate can be minimised in most assays by using AppliCoat Plate Stabiliser. Such effects depend on the used antibodies, but when ELISA are validated (e.g. according to “Guidance for Industry: Bioanalytical Method Validation”, FDA, 2001) or according to other validation strategies, the difference can be measured in many assays.The positive effects of AppliCoat Plate Stabiliser are shown in Fig. 1. A sandwich ELISA with a monoclonal antibody has been

Keywords

Immunoassays


ELISA Plates

Cross-reactivity

Interfering effects

No.4Improving quality of ELISA

AppliCationsThe Colorada product line is a selection of the newest fluorescent labels available today. Dye structures were carefully chosen to provide top-notch brightness perfor-mances, improving bioanalytical assays to the highest level. Eight different basic structures with precise absorption and emission maxima were selected to cover the full working range of wavelengths, starting from 400 nm up to 850 nm. The Colorada structures are based on 4 different chemical classes: coumarin, dipyrromethene borondifluoride, acenaphthopyrrole and cyanine

Coumarin dyes are chemically and photochemically robust, relatively small dyes with good absorption coefficients in the near UV / blue spectral region. Dipyrrometheneboron difluorides (BDPs) are chemically and photochemically robust, and pH insensitive. They have high absorption coefficients and excellent fluorescence quantum yields. Trimethine cyanine dyes have very high absorption coefficients, and are available in anionic (A), cationic (c) and zwitterionic (Z) form. 8-Oxo-8H-cyclopenta[a]acenaphthylene-7-carbonitriles (Acenaphthopyrrole dyes) are chemically and photochemically robust, pH insensitive dyes with high absorption coefficients and fluorescence quantum yields. Pentamethine cyanine dyes have extremely high absorption coefficients, good quantum yields of fluorescence and are available in anionic (A), cationic (C) and zwitterionic (Z) form. Heptamethine indocyanine dyes have extremely high absorption coefficients and good quan-tum yields of fluorescence in the near infrared (NIR) spectral region. They are available in anionic (A), and cationic (C).The dyes with a Large Stokes Shift (Colorada 670 LSS and Colorada 690 LSS) belong to the chemical class of heptamethine indocyanines as well. These dyes have high absorption coefficients in the far red and good quantum yields of fluorescence in the near infrared (NIR) spectral region, with a large stoke shift (> 100 nm).To meet the need of labeling the broadest range of target molecules each fluorescent compound is available with many dif-ferent reactive groups (e.g. the commonly used amine reactive and thiol reactive groups) while retaining the photophysical features of the parent dye. The reactive groups include carboxylic acid (- COOH), active ester (- COOX, NHS ester), dichloro-triazine, aliphatic amine (- NH ), azide (- N ), iodoacetamide and 2-pyridyldisulfideamine (PDA), the latter being very useful for labeling -SH in peptides or proteins.Furthermore, a new level of chemistry has been developed to provide the same fluorescent label with different charge values (anionic, cationic, zwitterionic and neutral). It is thus easy to find the ideal product among the Colorada series starting from the desired absorption/emission peak maximum and subsequently decide both the reactivity and charge properties.

Keywords

high absorption coefficients

high fluorescence quantum yields

wavelengths from 400–850 nm

coumarin

BDP

acenaphthopyrrole

cyanine

No.8Colorada: A New Generation Of Fluorescent Labels

AppliCationsEnhanced ChemiLuminescence Detection Kits for Horseradish Peroxidase in Western / Southern / Northern Blotting

The peroxidase-catalyzed oxidation of luminol produces a weak flash of light at 425 nm. The incorporation of a so-called enhancer into the buffer forces the flash signal into a glow and greatly improves the analytical characteristics of the reaction in terms of increased signal intensity and duration [1,2]. Typical enhancer compounds are substituted phenols, the most popular being p-iodophenol and p-hydroxycou-maric acid.

The enhanced, HRP catalyzed oxidation of luminol is a complex, multi-step reaction. While enhancers are useful in impro-ving enzyme turnover and increasing the equilibrium concentration of a key intermediate, the luminol radical anion, recent work [3] has shown that, by addition of a suitable catalyst, a further, large increase in light output is obtained:

As a result of this breakthrough, the family of AppliChem‘s CheLuminate-HRP chemiluminescent substrates has been developed, with specific formulations to meet the various requirements for immunoblotting (see table below for selecting the optimal substrate for your application).

Outstanding Sensitivity – The intense light output generated by the CheLuminate-HRP substrates translates into a corresponding improvement in sensitivity. Even the economical CheLuminate-HRP PicoDetect Extended formulation pro-vides a sensitivity level at least eight times higher than with phenolic enhancers, down to the low picogram (10 ) range. Detection limits are lowered further with CheLuminate-HRP FemtoDetect, to mid-femtogram (10 ), while the ultimate level in sensitivity, is achieved with CheLuminate-HRP FemtoDetect Plus, which is designed to provide femtogram (10 )- level detection.Long Signal Duration – All substrates exhibit long light emission. However, this feature has been specifically maximized in the formulation developed for the CheLuminate-HRP FemtoDetect substrate, which offers a 24-hour light emission – at least ten times longer than with standard phenolic enhancer substrates.

Keywords

Luminol

Redox Mediator

Enhancer

Long light emission

No.9CheLuminate: Improved Chemiluminescence

AppliCationsThe BARN PCR Blunt Cloning Systems -1 and -2 are based on the disruption of toxic barnase gene expression by cloning of PCR fragments into the multiple cloning site (MCS) of pBARN vectors. E.coli cells that contain non-recombinant vector are killed upon plating. Therefore, the blue/white screening is not required for this system. In contrast to other positive cloning systems BARN-1/-2 are not restricted to the use of special E.coli strains nor to a few restriction sites for the ligation of inserts only. In summary, pBARN cloning vectors combine advantages of positive cloning systems without the known disadvantages of other cloning systems available.

IntroductionMany cloning vectors in use today employ a blue/white screening approach to identify successful fragment insertions. DNA fragments (such as PCR products or pieces of a genome) are inserted into a multiple cloning site (MCS) within a plasmid DNA vector. Then, transformed competent E.coli cells are grown in the presence of X-gal. If the insertion was successful, the bacterial colony will be white; if not, the colony will appear in blue color. This traditional method, developed in the late seventies, has since been honed and simplified. However, it still has two major drawbacks: low efficiency of blue/white selection and high costs per cloning reaction. In addition, false-positive clones are frequently detected, making necessary sub-cloning and further time consuming investigation of candidate colonies, i.e. performing analytical restriction enzyme digest or sequencing.

To facilitate more simple and efficient high-throughput cloning several positive selection cloning systems have been developed (Table 1.). The principle of positive selection cloning is rather straightforward. Ligation of a PCR fragment into the MCS of a positive selection vector disrupts the expression/activity of the toxic gene, permitting growth of positive recom-binants (up to 100 %) upon transformation only. E.coli cells that contain non-recombinant vectors are killed upon plating. Disadvantages of known cloning systems that also employ positive selection of recombinants include: (i) The position of the poly-linker (MCS) in the middle of the toxic gene. This results in limitations regarding the choice of restriction enzymes as well as limi tations of using ‘universal sequencing primers’. (ii) Limitations for the bacterial strains to be used. (iii) Limited size of the DNA inserts. (iv) In some cases handling is cumbersome.

The pBARN PCR Blunt Cloning System is an easy-to-use, fast and highly efficient positive cloning system of blunt or blunted PCR fragments. Fragments generated by either Taq DNA polymerases or other DNA polymerases without proof-reading activity require a filling-in reaction for optimal cloning efficiency. The pBARN PCR Blunt Cloning System is based on the toxic barnase gene product, a small, highly active ribonuclease from Bacillus amyloliquefaciens (Yazynin et al. 1996; Yazynin et al., 1999; Fig. 1). pBARN-1 and -2 PCR Blunt Cloning Vectors (3.4 kb and 3.2 kb, respectively), the main component of these systems, allow direct selection of recombinants via disruption of the lethal barnase gene, once expressed. The vector contains the barnase gene under the control of the LacZ promoter, N-terminally fused to an especially designed sequence, including the multiple

Keywords

Molecular Cloning VectorPCR Blunt Cloning

Positive Selection

Barnase

No.11pBARN Cloning Vectors – Positive Selection of Recombinants AppliCations

Cell proliferation assays are widely used in cell biology for the study of growth factors, cytokines or media components. They are also applied in the screening of cytotoxic agents and lymphocyte activation. In order to determine the number of viable cells Cell Proliferation Kit XTT employs 2,3-Bis-(2-methoxy-4-nitro- 5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT). Only in living cells mitochondria are capable to reduce XTT to form an orange colored water soluble dye. Therefore, the concentration of the dye is proportional to the number of metabolically active cells.

The need for a reliable, sensitive and quantitative assay that would enable analysis of a large number of samples led to the development of methods, such as incorporation of radioactively labeled H-thymidine into DNA or the use of 5-Bromo-2‘-deoxyuridine (BrdU) as a substitute for radioactive thymidine to label DNA in living cells.The above methods have a number of disadvantages, including: use of radioactive materials and relatively complex techniques. The use of tetrazolium salts, such as MTT, commenced in the 1950s, is based on the fact that living cells reduce tetrazolium salts into colored formazan compounds.

Keywords

XTT assay

cytotoxicity testing

non radioactive assay

quantitating and viability testing of cells

No.12Cell Proliferation Assay XTT

AppliCationsIn analytical laboratories, it is common practice to „seal“ bottles of HPLC solvents simply with a perforated aluminium foil or plastic cap. Connecting tubes are inserted into the bottles through the holes in the foil/cap without any additional sealing. However such basic covers do not seal the solvent containers efficiently. On entering an HPLC laboratory, one can often already smell the solvent vapors. This uncontrolled evaporation of solvents is not only a potential health hazard but also the loss of solvent by evaporation interferes with the quality and reproducibility of HPLC runs. We present here quantitative data comparing the efficiency of various sealing methods, including Applichem’s SafetyFirstCaps for HPLC.

To test for the reproducibility of the retention time and the loss of solvent by evaporation, tests were performed over a period of 31 days. The separation of three PAHs (Polycyclic Aromatic Hydrocarbons) with solvents, stored in either tightly closed bottles (SafetyFirst Caps) or bottles closed with caps with holes of different diameters, was compared. The results of the tests were dramatic.

Test Conditions

Bottle A This Bottle was closed with a SafetyFirst Cap, fitting precisely the standard GL45 glass bottle thread used.Bottle B This bottle was tightly closed with the GL45-cap provided including a Teflon-foil seal.Bottle C This bottle was closed with a cap having a 10-mm hole leaving an opening of an area of approximately 0.785 cm .Bottle D This bottle was closed with a cap having 3 holes, 3 mm each, leaving an opening of an area of approximately 0.212 cm .

Procedure

• At the start of the test, all 4 bottles were filled with the same mixture of Water + Methanol = 20 + 80 (w/w).• Using Bottle B as a reference, chromatograms of a mixture of the three PAHs (Naphthalene, Pyrene, Chrysene), were compared with the reference.• After the first measurement, all bottles were kept at room temperature under a fume hood with a gentle air flow for 31 days.HPLC-System HITACHI LaChrom Elite system with Diode Array Detector under control of the EZChrom Elite Software. Isocratic pump conditions with premixed mobile phase.HPLC-Column Purospher RP-18e (5µ), 125 x 4 mm

Keywords

health protection

reduction of contaminant concentrations

constant HPLC-retention time

save solvents

No.10SafetyFirst Caps: Safety-Systems for HPLC

AppliCationsSeparateIT gels represent a novel gel matrix for DNA electrophoresis. Gel polymers are arranged in a conceptually different way, in accordance with a new theoretical model of gel electrophoresis. SeparateIT gels selectively retard the migration of large molecules, so that DNA bands remain sharp but are more spread out relative to each other. Thanks to this increased spacing, resolving power of SeparateIT gels is at least twice higher compared to resolving power of any other gels, including polyacrylamide gels.

The extraordinary resolving power of SeparateIT gels results from addition of a special polymer to a polymerizing solution containing a monomer and a cross-linker. While the gels with SeparateIT-like properties could be prepared with several different polymers, chemical composition and molecular weight of the polymer required a careful optimization for each particular monomer/cross-linker combination.In order to satisfy numerous requests from researchers who wish to improve resolving power of their acrylamide based gels, AppliChem now provides SeparateIT Polymer Solution. This polymer has been optimized for the gels with a ratio of acrylamide to N,N-methylene-bisacrylamide of 29 : 1. The polymer is not optimized for denaturing, urea-containing polyacrylamide gels.

SeparateIT Polymer Solution comes as a 10X solution. It should be mixed with a buffered solution of acrylamide and Bis prior to addition of TEMED and ammonium persulfate. Gels with SeparateIT Polymer Solution are prepared in the same way as any regular polyacrylamide gels. Likewise, the electrophoresis, gel staining and recording are carried out as usual. The increased resolving power of SeparateIT gels enables full separation of closely spaced bands on short gels. For example, a pair of fragments differing by 4 bp is usually resolved on less than 4 cm of gel length. Two DNA fragments in the 70 –150 bp range that differ by 1 bp can be separated on SeparateIT gels that are 8 cm long.We recommend that Mini gel cassettes, which are 8 or 10 cm long, are used for casting acrylamide-Bis gels with SeparateIT Polymer Solution. Such relatively short gels will be appropriate for the majority of demanding separations which have previously required 20 –30 cm long polyacrylamide gels. The use of shorter gels is beneficial for several reasons, including easier gel preparation and handling, lower cost of the gel materials, faster electrophoresis runs, and lower consumption of gel staining reagents. In addition, a lower amount of sample DNA needs to be loaded on a gel with a high resolving power compared to a gel with a low resolving power. This is the case because DNA bands that migrate a short distance remain sharper than the bands that migrate a long distance. It is always advantageous when closely spaced bands separate after migrating just a few centimetres.

Keywords

improved resolution

Polyacrylamide Gel Electrophoresis

SeparateIT Polymer Solution

No.7Improving Separation During Electrophoresis

Keywords

improved resolution

Polyacrylamide Gel Electrophoresis

SeparateIT Polymer Solution

AppliCationsSize-exclusion chromatography (SEC) is a popular method to separate biomolecules based on their size. Primarily, it is applied to the separation of biopolymers such as proteins and nucleic acids, i.e. water-soluble polymers. This system is also called gel filtration, typically with beads of dextran or agarose serving as gel matrix. Smaller molecules pass significantly slower through the column than larger molecules. Not to be mixed up with gel electrophoresis, there are big differences in terms of the separation principle. SEC does not require electric current and the sieving effect will not separate small molecules first.

It is indeed correct that smaller molecules pass more slowly through the matrix than larger molecules. This is due to the longer path the smaller molecules must travel. The longer path arises from the pores of the beads. The smaller molecules can enter the pores and go inside the beads. Entering the pores creates a longer path for the smaller molecules. There is a relationship between the delay this causes and the molecular size. The larger molecules can not enter the pores and therefore have a shorter path. The larger molecules therefore travel faster through the column than smaller molecules.Advantages of SEC/Gel FiltrationSome important features of the technique have to be mentioned. First, the separation of the large molecules from the small ones is very effective and is performed under mild conditions, does mean biomolecules keep their biological activity. The volume of the eluate may be kept small and therefore, the techniques is suited to concentrate samples. The MatrixAppliChem‘s size-exclusion chromatography columns are packed with AppliXchange-G25 M, a beaded composite material composed partially of polymerized dextran. It exhibits high selectivity, high resolution and chemical stability. Molecules purified with AppliXchange-G25 M are separated according to size. The chemical interaction of gel matrix and molecules to be separated is negligible. No absorption takes place. Buffer and pH effects on resolution are minimal. The size exclusion cut-off for AppliXchange-G25 M is set at 10 kD for proteins and 10 bp for nucleic acids. Purified biomolecules are not significantly diluted when processed using AppliXchange-G25 M.The number “25” correlates to the “water regain” of the matrix. A grade “25” means that the dry matrix will take up 2.5 times its weight of water (1 g AppliXchange-G25 M absorbs 2.5 g water). This is a water regain of 2.5. Likewise, a grade of “50” gives a water regain of 5 grams (1 g AppliXchange-50 plus 5.0 g water.) The ability to absorb water is dependent on the extent of cross-linking. Tighter cross-linking limits the ability of the beads to swell and as a consequence the water regain is lower. Less cross-linking gives a more flexible bead and allows additional swelling. The extent of cross-linking has been optimized for a particular water regain. The water regain/extent of cross-linking affects the separation properties of the gels. Larger water regain/less cross-linking results in larger pores, which in turn retains larger molecules. Less water regain/more cross-linking results in smaller pores, which in turn retains only the smallest molecules. The separation efficiency is

Keywords

Gel filtration matrix

cross-linked dextran beads

Desalting

Buffer exchange

Nucleic acid/ protein purification

No.6Size-Exclusion Chromatography for purification of biomolecules

Keywords

Immunoassays


ELISA Plates

Cross-reactivity

Interfering effects

AppliCations

AppliCationsMany biochemical processes are markedly impaired by even small changes in the con centrations of free H ions. It is therefore usually necessary to stabilise the H concentration in vitro by adding a suitable buffer to the medium, without, however, affecting the functioning of the system under investigation. A buffer keeps the pH of a solution constant by taking up protons that are released during reactions, or by r eleasing protons when they are consumed by reactions.

This handout summarizes the most commonly used buffer substances and respec-tive physical and chemical properties.

Practical Tips – Preparing Buffer SolutionsRecommendations for the setting of the pH value of a buffer and storage conditions1. TemperatureDepending on the buffer substance, its pH may vary with temperature. It is therefore advisable, as far as possible, to set the pH at the working temperature to be used for the investigation. For instance the physiological pH value for most mammalian cells at 37°C is between 7.0 and 7.5. The temperature dependence of a buffer system is expressed as d(pK )/dT, which describes the change of the pK at an increase of temperature by 1°C.

2. Titration(i) Generally, the pH value is set using NaOH/KOH or HCl. Slow addition of a strong acid or base whilst stirring vigorously avoids local high concentrations of H or OH ions. If this is not done, the buffer substances may undergo chemical changes that inactivate them or modify them so that they have an inhibitory action (Ellis & Morrison 1982). (ii) Under stirring COdissolves in the solution. Stir solutions gently for precise measurements of the pH value. (iii) If a buffer is available in the protonised form (acid) and the non-protonised form (base), the pH value can also be set by mixing the two substances. (iv) Setting of the ionic strength of a buffer solution (if necessary) should be done in the same way as the setting of the pH value when selecting the electrolyte, since this increases depending on the electrolyte used. (v) If other components are added to the buffer (e.g. EDTA, DTT, Mg , b-Mercapto ethanol) changes in the pH should also be considered and pH should be retested. (vi) In the presence of divalent metal ions carbonate or phosphate buffers may form precipitates .

3. How can microbial contamination of buffer solutions be prevented?(i) Sterilization by filtration through a 0.22 µm filter unit or by autoclaving. (ii) Addition of 0.02 % (3 mM) sodium azide. (iii) Storage at +4°C. (iv) High-concen tration stock solutions.

Keywords

chemical properties

usefull pH range

buffer preparation

No.2Biological Buffers

AppliCationsAdvanced experiments in gene technology demonstrate that even small amounts of free DNA molecules are sufficient to cause infections, recombination or biological transformation [1,2]. The complete decontamination of equipment and surfaces from any DNA molecules is important for biological containment and safety, as well as preventing artifacts in PCR amplification experiments. Using new methods that detect extremely low levels of DNA molecules, we investigated the molecular mechanism of action of various commercially available DNA decontamination reagents. We found that when using high concentrations of DNA and short incubation times, none of the conventional reagents destroyed DNA molecules efficiently, despite their corrosive or even toxic properties.

All or Nothing at AllDestruction and elimination of nucleic acids depends to this day, on bleach, a corrosive and toxic substance. To address this issue, AppliChem partnered up with multiBIND Cologne, to develop a new and unique nucleic acid decontamination technology: the ExitusPlus™ family of products, comprising DNA-ExitusPlus™, and Autoclave-ExitusPlus™. Comparing DNA-ExitusPlus™ to conventional products, we can demonstrate that it is fast and efficient in destroying nucleic acids without harmful or toxic effects on lab workers, equipment and the environment. Most decontamination reagents are based on several molecular principles for the destruction or inactivation of genetic material: Modification and denaturation can mask, but do not destroy the genetic information encoded in DNA strands and there is the risk that they may be chemically re-activated. Thus, safe and complete DNA decontamination depends on the degradation of DNA into very small fragments. Figures 1 and 2 show the results of comparing the fragmentation process produced by the novel DNA-ExitusPlus™ with conventional reagents. Using highly sensitive detection assays, it is seen that an efficient and total fragmentation was only obtained with DNA-ExitusPlus™, whereas only partially degraded DNA pieces, some of which contained complete genetic information, were found in the decontamination products that relied on modi-fication or denaturation methodology.

Sequence-Independent Degradation of DNAOnly Applichem’s DNA-ExitusPlus™ is capable of achieving rapid and efficient degradation of nucleic acids, because its unique method of action is based on chemical and not enzymatic activity. Therefore, its effects on fragmentation are totally independent of the size and sequence of the DNA fragments. Larger plasmids require a longer incubation time than smaller ones (e.g. primers). Assuming a theoretical nicking activity of 100,000 nicks per minute, all DNA fragments will be destroyed, after several minutes, regardless of their size. Smaller fragments will disappear before the larger ones. Applying this theory to a test molecule (ccc form, 6 kb plasmid) only a small fraction of fragments with 200 to 500 bp in size will remain after 5 minutes. The nicks will be introduced statistically at any site, leaving not a single class of fragments. Remaining fragments are fully destroyed after 10 minutes.

Keywords

Nucleic acid decontamination

DNA degradation test

Autoclaving DNA

PCR test

No.1Nucleic Acid Decontamination with The ExitusPlus™ Technology

Orders by fax 06151/93 57 11 by eMail [email protected]

Get your free copy!

AppliChem provides background information or published application notes for some selected products.


AppliChem’s Blocking Buffer I, is a reliable result achieved. At first view, what appears as an unimposing effect, ultimately influences the significance of all subsequent tests. In most cases, ELISAs, Western Blots or other immuno assays simply serve as tools to verify or falsify a hypothesis of the whole research project. Those who use unreliable tools, as shown for the BSAblocked

blocking

Blocking in immunoassays is an important step, i.e. the saturation of free binding sites on surfaces of ELISA wells or Western Blot membranes. Noncomplete blocking will be cursed with high background and either useless or nonmeaningful results. Therefore, several very different methods for blocking were developed and became established especially proteinbased blockers containing casein or BSA. The caseinbased blockers show an unsurpassing efficiency, but only when they are prepared using casein which has undergone severalfold cleavage. Whenever reproducible results with a low standard deviation are of importance, no serious alternative to casein exists. Unfortunately, preparation is complex making this product more expensive. Now, we present a new alternative, Blocking Buffer II EGrade, combining quality and economy.

State of the artIf samples are rare, expensive, or the results have to be reliable, then there exists no alternative to the highly purified caseinbased blocking reagents (e.g. Applichem's Blocking Buffer I). The only option available is based on ultrapure BSA and in many test systems, efficiency is comparable. Differences appear in terms of standard deviations when real samples are measured (determined as coefficient of variation (CV) for multiple measurements). These differences are mainly due to differences in molecular size of the blocking agent. The relatively large BSA leaves gaps on the support surface which will be covered by smaller molecules coming from the sample. Most of these substances do not influence the results. Some do interact with e.g. detection antibodies or the label such as peroxidase, phosphatase or fluorescent dyes. As soon as interactions occur, they lead to erroneous results. The difficulty is that one cannot predict when it will happen, how strong the interference will be and whether deviations will shift results up or down. You can identify such interferences simply by observing high standard deviations, i.e. high CVs. Looking at the CV as shown in fig. 1, the problem is obvious. Figure 1 shows a sandwich ELISA, which seemed to work well by including a BSAbased blocking reagent (Blocking Buffer III BSA). But CV’s tell something different. Upon first glance, the assay looks good but the results are unreliable. Not until running the same assay using

Dr.WolframH.Marx,AppliChem

Comparing Blocking ReagentsQuality and economy now combined in a novel blocker


ELISA, must be prepared that their results will be questionable and, in the present case, doubts are justified.

There is another problem arising from time to time that must not be overlooked. Over the years it has been common practice to couple small antigenes, socalled haptens, to BSA as carrier molecules to generate antibodies. It works well, is reliable and simple. The problem is that a large number of commercially available antibodies as well as antibodies generated by and exchanged between scientists not only bind the hapten with significant affinity but also BSA. This wouldn't be a problem if the producers of such antibodies would mention the method of immunization. In those cases, one would be aware of the potential interference and would not choose a BSAbased blocking reagent. Unfortunately, the exact opposite is reality and research antibodies do not include this essential information. Nevertheless, in most cases BSA is a very good blo cking reagent. It is cheaper than optimized caseinbased blocker, even if the “BSA" purification is laborious.

The big disadvantages of good proteinbased blo ckers is the high costs and the search for alternatives has resulted in several varieties. Gelatin from fish or nonfat dried milk are just two to mention. In some laboratories and assays they meet the requirements and, in many other assays they constantly fail. Due to the uncontrollable quality of these raw materials, these blocking reagents are not used in medical research, e.g. in pharma assays, preclinical or clinical studies or in diagnostics, since reproducibility is not given. Reproducibility of an assay depends on constant quality of the raw materials.

Alternatives wantedSince the desired alternative needs to be of high, reproducible quality but at low cost, synthetic blockers were investigated and Tween® represents the first, best known and simplest solution. Other synthetic blockers are also availavle at low prices as well. While consistency of raw materials may be optimal, one problem couldn't be solved: reproducibility of results thereby limiting their use. Considering that the total costs of blocking are just a small part of the total costs of an assay, it is obvious why very few scientists would trade quality for marginal savings.

Now, AppliChem presents THE new alternative for efficient and costeffective blocking in immunoassays. Blocking Buffer II EGrade is manufactured to DIN ISO 9001:2000 quality requirements, free of any serum proteins, and peptidebased. Since the synthesis of the peptides is controlled, blocking is uniform and reproducible from batch to batch. The costs are comparably lower than for proteinbased blockers, while quality is acceptable – in contrast to synthetic blocker.

Fig. 1Comparisonofthecoefficientofvariation(CV)ofanELISAwithbloodsamples.TheELISAwasperformedoncewithBlockingBufferIandoncewithBlockingBufferIIIBSAunderidenticalconditions.TodeterminetheCVacompleteELISAplatewasmeasured(n=96).

coef

fici

ent o

f var

iati

on [

%]

Blocking Buffer I Blocking Buffer III BSA

Fig. 2aOD-valuesforblockingovernightwithdifferentblockingreagents.Thisisamodifiedanalyte-freeassayoriginallydevelopedbySteinitz&Baraz(2000),measuringtheinfluenceofblockingwithoutnegativeeffectsofrealsamples.

Fig. 3Graphofthecoefficientofvariation(CV)oftheassayfromfig. 2.ThelowertheCV,themorereliabletheassay.Inindustrialpractice,CVsof<15%forELISAswithrealsamplesareneeded(e.g.accordingtoFDAguidelines).PleasenotethattheCVsshownhereonlyrepresenteffectsofblockingreagents,sincetheassaydon'tincludesanalytesandothernegativeeffects.

Fig. 2bImageoftheELISAplatedescribedin fig. 2a.Theleftcolumnisthecontrolwithoutblocking(K-).Theorderofblockingreagentsisasinfig. 2aBlockingBufferI(BBI),BlockingBufferIIIBSA(BBIII),BlockingBufferIIEGrade(BBII)andsyntheticblocker(SyB).

K- BB I BB III BB II SyB


CV [

%]

OD

450

[nm

]


Blocking Buffer II Egrade

synthetic Blocker


Blocking Buffer II Egrade

synthetic Blocker

Conclusion

For many assays in LifeSciences, AppliChem offers an alternative to high cost proteinbased blocking reagents. Blocking Buffer II EGrade has the potential to combine economy and quality.

Literatur [1] Steinitz, M. & Baraz, L. (2000) J. Immunol. Methods 238,

143-150. A rapid method for estimating the binding of

ligands to ELISA microwells.

[2] Steinitz, M. (2000) Anal. Biochem. 282, 232-238.

Quantitation of the Blocking Effect of Tween® 20 and

Bovine Serum Albumin in ELISA Mircrowells.

Analyte-independent comparison

Is it possible to determine the real blocking efficiency and quality of a new blocking reagent? According to the methods of Steinitz & Baraz (2000) and Steinitz (2000), a test system was established with the following changes: peroxidase was used instead of phosphatase as reporter enzyme, eight determinations instead of double values, and additional determination of the coefficient of variation (CV).

The test included Blocking Buffer I, highly purified and multiply digested casein, Blocking Buffer III BSA based on BSA, the novel Blocking Buffer II EGrade, as well as a commercial, synthetic blocking reagent.

The surface of the ELISA wells were blocked by the different blocking reagents and incubated with a detection antibody (analog to Steinitz (2000), but using peroxi dase) without any additional additives. After washing, the staining reaction was carried out. The lowerthe optical density (OD), the better the blocking efficiency.

Figure 2 shows blocking overnight. All variants do block. The blocking efficiency of the proteinbased blocking reagent is superior, good for Blocking Buffer II EGrade and also good for the synthetic product. The graph in fig. 2a shows the ODs, fig. 2b the corresponding image of the blocked ELISA plate. The left column served as an unblocked control.

The coefficient of variation of the same experiment is presented in fig. 3. Here, the wheat separates from the chaff. At first view, the synthetic solution looks promising. Even though a simple, artificial test system without any interfering substances was used, the CV value of 25 % is extremely high. Such a CV is transferred to the total CV of an assay according to the error propagation of each separate error of the assay. It is questionable whether a reproducible assay may be established with such a synthetic blocking reagent. The advantage of the novel Blocking Buffer II EGrade is clear. Blo cking is highly reproducible with a constant CV, identical to the proteinbased reagents.

Combining the results shown in fig. 2 and 3 allows one to draw the conclusion that in those assays, where Blocking Buffer II EGrade gives a low background, reproducabillity is seen also. This is an advantage over other alternatives like the fish gelatin, milk powder or synthetic blocking reagents. Blocking Buffer II EGrade combines economy and reproducibility for various assays, and may be regarded as a suitable alternative to the proteinbased blocking reagents. The comparison according to Steinitz also proves the unique position of proteinbased blockers for sensitive applications like pharma assays, clinical studies or other assays including valuable samples.



Using ready-to-use ELISA kits from manuf-acturers is easy and convenient. Some-times however, home-made ELISA are required, because there is no kit available with the right antibodies or characteri-stics, such as limits of detection are not appropriate. Ready-to-use ELISA kits from good suppliers may be stored for two years at 4°C without any problem. With home-made ELISA kits it is a completely different story. For any new measurement one has to coat a new plate, because after storage of some days the plates don’t perform as well as before.

Why is there such a great difference in the suitability for storage between home-made ELISA and commercial ELISA kits?The reason is that in commercial ELISA kit production the plates are not only blocked after coating, but also stabilised. This easy to perform process has been an industry standard for thirty years. For stabilisation of a plate one has to incubate with a coating stabiliser solution. It is just as simple as a “second blocking step”. But there were no such high quality stabiliser solutions freely available in low volumes for use in research lab until now. AppliChem now offers a product for use in every research lab in volumes starting as small as 50 ml, which is called the AppliCoat Plate Stabiliser (Cat. No. A7708). This stabiliser solution is easy-to-use and has a great advantage compared to al-most any stabiliser used in industry. It gives better storage stability for coated anti bodies and antigens than most other products do. And there is a second benefit.

Two benefits with one solutionWhen antibodies are coated onto ELISA plates, most of the antibodies are not active. When the antibodies (or any proteins) come into close contact to the plastics surface of the ELISA plate, conformational changes can occur due to surface-protein interactions. The result is that most antibodies coated on a plate are unfolded or in active. Only around 2–8 % of all coated antibodies remain active and can bind to analytes and this is greatly variable depending on the surface characteristics of the ELISA plate, which can really differ from batch to batch or even from well to well.These differences from well to well can affect the variability of an assay, be-cause the antibodies can be affected. If there was a way of refolding antibodies and of preserving antibodies from conformational changes during storage, this could help to decrease such variabilities in assay performance. This is a key benefit of AppliCoat Plate Stabiliser. It assists antibodies and coated pro-teins to refold and then to preserve active conformation over a long time. Thus it has two benefits: 1. Refolding of antibody conformation of some of the

Improving quality & stability of ELISA

coated antibodies and 2. Preserving correct conformation during storage. These benefits are used for production of high-quality ELISA kits as well as in research applications now. Even with AppliCoat Plate Stabiliser the percentage of active antibodies will still be in the range of 2-8 %. But the great difference is that the variability from well to well and from plate to plate can be mini-mised in most assays by using AppliCoat Plate Stabiliser. Such effects depend on the used antibodies, but when ELISA are validated (e.g. according to “Guidance for Industry: Bioanalytical Method Validation”, FDA, 2001) or according to other validation strategies, the difference can be measured in many assays.The positive effects of AppliCoat Plate Stabiliser are shown in Fig. 1. A sand-wich ELISA with a monoclonal antibody has been done. This monoclonal AB lost its binding activity when coated on a plate and stabilised only with BSA without applying AppliCoat Plate Stabiliser. Figure 1 shows a high tempera-ture “stress test” at 37°C for 84 days after application of the stabiliser solution. The real OD signal is shown without any normalisation, which could poten-tially falsify interpretation of the results. One can clearly see the better binding activity when AppliCoat Plate Stabiliser is used. If this plate was stored dry at 4°C the test would correlate to around 2 years of storage.

An improved workflow for most labsThis advantage of a prolonged storage time can help to set up a completely new workflow in research labs. Whilst nowadays it is best practise to produce a new and fresh ELISA plate whenever some measurements with a home-made ELISA are done, this can change. One can easily coat, block and stabi-lise as much plates as needed for the next 3 to 6 months. Those plates are dried in an incubator at 20°C or 30°C for 60 to 120 minutes, the plates can be stored in the fridge for several months. Those plates can be used from time to time for measurements. All those plates are therefore produced in one batch in your lab minimising plate-to-plate variations coming from differences in plate production.Thus the new workflow in labs has three great advantages:1. Less work, because coating and blocking of many plates can be done in one

run instead of doing this work for any new plate again and again.2. Lower variations in measurements from plate to plate as described above.3. Saving time and potentially improving the quality of the results of a lab.

Industrial technology now available even for small research labsAccording to the needs of research labs AppliCoat Plate Stabiliser (A7708) is available in volumes of 50 ml, 125 ml and 500 ml. It can be used in combi-nation with standard blocking solutions like casein (e.g. Blocking Buffer I, A7099) or BSA based solution (e.g. Blocking Buffer III BSA, A7252) and other blockers. For any researcher this industrial stabilisation technology is made available by AppliChem. Even small labs can make use of it. Test it for yourself!


How to use AppliCoat Plate Stabiliser?

Figure 3. CalibrationcurvesofanELISAareshown,whichwaseither

blockedonlywithBSA(red)orblockedwithBSAandadditionallysta-

bilizedwithAppliCoatPlateStabilizer(blue).Calibrationcurvesatthe

startingdayareidentical.Thesensitivityoftheassaywithoutstabilization

decreasesrapidlyduringstorageat37°C.Duringthisstresstestthe

ELISAafterstabilizationdoesnotshowadecreaseinsensitivityevenafter

84daysofincubation.

Figure 1. Coating,blockingandstabilizationofELISAplatesina3step

andina2stepprocess.Thesolutionsareadded,incubatedandremoved

again.Thentheplatesaredriedandsealedinplasticoraluminiumfoilfor

drylong-termstorage

Figure 4. BindingactivityofanELISAatthestartingdayandafterstorage

for84daysat37°Cisshown.Infig.3thecompletecalibrationcurvesare

shown(onlyforBSAblocking).Hereweshowthemeasurementsat

40ng/ml.ActivationofbindingcapacitybyAppliCoatPlateStabilizercan

beseenquiteclearly.Itisalsoshown,thatAppliCoatPlateStabilizercan

becombinedwithdifferentblockerswithoutanyproblems.

Figure1.showsthesimplethreestepproductionprocess,whichcanbeusedforkitproductionaswellasforhomemadeELISA.

1. Coating as routinely.2.Blockingasroutinely.3.Platesarewashed3timeswith200-300µlPBSoraWashingBufferwithoutdetergents(e.g.WashingBufferTrisT-(10x)ordernumberA7137)

4.Add200µlAppliCoatPlateStabiliserandincubatefor30-90minutesatap-prox.20-30°C

5.RemoveAppliCoatPlateStabiliserbysuction.Incubatetheplatesat37°Cuntildry.Thisneedsaround60to120minutesdependingonincubatortype,numberofplatesintheincubatorandspeedofaircirculationintheincuba-tor.

6.Storetheplatewithalidinthefridgefor3to6months.Alternativelyplatescanbe sealedwithplastic or aluminium foil underdryness.Whenplatesarestoreddry,theshelflifewillbearound1to3years.

Achievableshelflifecandifferfromantibodytoantibody.Theusedplates,coatingconcentration,proceduresandbuffershaveadditional impactonachievablestability.Thusifplatesshouldbestoredformorethan6months,astresstestat37°Cor45°Cforstorageshouldbedonewiththeplatestoeasilymeasuretheshelflifeoftheplateinashorttime.

Figure 2. Stresstestat45°Cwithamonoclonalantibody.Thisantibody

showedevenafterstabilizationwithcarbohydratecontainingsolutions

ofthefirstgenerationnobindingfunctionalityafter24hours(data

notshown).ThestabilizersofthesecondgenerationA,BandCshow

acceptablestabilization.AfterstabilizationwithAppliCoatPlateStabilizer,

amodernstabilizerofthethirdgeneration,thisunstableantibodyshows

evenafter66daysofincubationat45°Cmorethan85%ofitsactivityand

thereforeanimpressivelyenhancedstability.


Sometimes, less is more!

For The Sake OfThe Environment

Many reagents prepared in biomedical research labs are ideal nutrient broths for unwanted germs (bacteria, fungi). To prevent their growth, reagents are either autoclaved, sterile filtered, or antibiotics / antimycotics or toxic substances are added. One of these additives is Thimerosal, a mercury-containing molecule which is dangerous for the environment. Our original immunoasssay buffer contained this chemical too, but now we have replaced it by the nontoxic ProClin® 300. For the sake of the environment.

For Your Safety

We would like to keep you healthy so that you stay our customer. FYI: Thimerosal is classified as toxic. The lethal dose (rat, s.c.) is 9 mg/kg, compared to ethidium bromide with a lethal dose (mouse, s.c.) of 110 mg/kg. In some countries, Thimerosal is a forbidden additive.

For Better Results

Changing the composition has no negative influence on the performance of the products. With CrossDown and all other immunoassay buffers you are even in a better mood and your immunoassays show a better quality.


products Sometimes, less is more!


Antibody Stabilizer-PBS A7148

Stabilization buffer for long-term storage of antibodies and proteins A7148,0050 50 ml A7148,0125 125 ml

pH value pH 7.4 ± 0.2Preservative contains 0.1 % ProClin®300Storage 2-8°C. Avoid freezing *Stability at 2-8°C: 2 years* Upon freezing, an insoluble precipitate is formed. The activity is not significantly reduced!

Instructions for useAntibody Stabilizer-PBS is made for long-term storage of proteins and antibodies at 2-8°C in solution.Components of Antibody Stabilizer conserve the structure of proteins and antibodies in solution. Thus proteins and antibodies are prevented from loosing functionality during storage. Usually, antibodies / proteins are diltued in Antibody Stabilizer-PBS at least by a factor of 1:20, but significant further dilution is possible. The typical concentration of antibodies during storage is 400 to 1000 ng/ml. Many antibodies may be stored even at concentrations as low as e.g. 80 ng/ml in Antibody Stabilizer-PBS, without significant loss of binding activity within the first two years. Such low concentrations do not require pre-dilutions before every single application.Immediately before use Antibody Stabilizer-PBS should be mixed thoroughly. Just dissolve proteins or antibodies with Antibody Stabilizer-PBS for storage in the refrigerator.You can also use Antibody Stabilizer-PBS for storage of coated ELISA plates. After blocking add Antibody Stabilizer-PBS to the coated plates. In this manner treated plates can be stored for a longer time in the refrigerator.Stability of proteins and antibodies differs significantly from case to case. Stability depends on characteristics and concentration of the used proteins and antibodies. The user has to test it therefore with its own proteins or antibodies.Antibody Stabilizer-PBS is ready-to-use.

Antibody Stabilizer-Tris A7135

Stabilization buffer for long-term storage of antibodies and proteins A7135,0050 50 ml A7135,0125 125 ml

pH value pH 7.2 ± 0.2Preservative contains 0.1 % ProClin®300Storage 2-8°C. Avoid freezing *Stability at 2-8°C: 2 years

Instructions for useAntibody Stabilizer-Tris is made for long-term storage of proteins and antibodies at 2-8°C in solution.Components of Antibody Stabilizer-Tris conserve the structure of proteins and antibodies. Thus proteins and antibodies are prevented from loosing functionality during storage. Usually, antibodies / proteins are diltued in Antibody Stabilizer-Tris at least by a factor of 1:20, but significant further dilution is possible. The typical concentration of antibodies during storage is 400 to 1000 ng/ml. Many antibodies may be stored even at concentrations as low as e.g. 80 ng/ml in Antibody Stabilizer-Tris, without significant loss of binding activity within the first two years. Such low concentrations do not require pre-dilutions before every single application.Immediately before use Antibody Stabilizer-Tris should be mixed thoroughly. Just dissolve proteins or antibodies with Antibody Stabilizer-Tris for storage in the refrigerator.You can also use Antibody Stabilizer-Tris for storage of coated ELISA plates. After blocking add Antibody Stabilizer to the coated plates. In this manner treated plates can be stored for a longer time in the refrigerator.Stability of proteins and antibodies differs significantly from case to case. Stability depends on characteristics and concentration of the used proteins and antibodies. The user has to test it therefore with its own proteins or antibodies.Antibody Stabilizer-Tris is ready-to-use.

ProClin® is a registered trade mark of Rohm and Haas Company.


AppliCoat Plate Stabilizer A7708

Preservative for long-term storage of coated surfaces in immunoassays A7708,0050 50 ml A7708,0125 125 ml A7708,0500 500 ml

pH value pH 6.5 ± 0.2Preservative contains 0.1 % ProClin®300Storage 2-8°C.Stability at 2-8°C: 1 year

Improving quality of ELISA measurements and enabling easier workflow in research labsAppliCoat Plate Stabilizer is a ready-to-use reagent for the preservation of immobilized antibodies and proteins. AppliCoat Plate Stabilizer is simply added to the coated microtiter plates, polystyrene beads or glass slides. AppliCoat Plate Stabilizer seals by forming a uniform stabili-zing layer over antibodies and antigens immobilized on the solid phase. This layer is distinguished by good solubility without affecting assays performed at a later date. It is used for long-term storage of precoated plates and other surfaces used in immunoassays.AppliCoat Plate Stabilizer is used directly after blocking and washing. AppliCoat Plate Stabilizer seals and stabilizes coated proteins and antibodies. In the case of strong background problems we recommend the use of AppliChem Blocking Buffer I (A7099) before sealing. Blocking Buffer I is characterized by a higher blocking efficiency than most other known blocking reagents.After incubation of the microtiter plate or solid phase with AppliCoat Plate Stabilizer, the plate can be stored either directly under moist conditions or after drying of the plate or solid phase. The shelf life of the coated molecules is substantially extended to typically 1 to 3 years when stored cool and dry. The assay buffer or the specimen can be added directly onto the sealed plate (solid phase) for use in the assay. An additional washing step is not necessary.AppliCoat Plate Stabilizer is free of proteins. The values for shelf life of sealed plates are to be used only as a guide. Longer shelf lives have been observed, but this may not be generalized for all assays. Therefore any assay has to be tested for its individual shelf life.

Figure 1: Hightemperature“stresstest”at37°Cfor84daysafterapplicationoftheplatestabilizersolution.ThetrueODsignalisshownwithoutanynormalisation,whichcouldpotentiallyfalsifyinterpretationoftheresults.OnecanclearlyseethebetterbindingactivitywhenAppliCoatPlateStabilizerisused.Whenthisplatewouldhavebeenstorednotat37°Cbutdryat4°Cthetestwouldcorre-latetoaround2yearsofstorage

Figure 2: ApplicationofAppliCoatPlateStabilizer.Stabilizationissimilartoa“secondblockingstep”.AfterthistheELISAplatesaredriedinanincu-batorandcanbestoredinafridgeforalongtime.

Instructions for use1. Apply commonly used coating and blocking pro-cedure for microtiter plate.2. After blocking: Wash 3 times with 200-300 µl PBS or Washing buffer without detergents (e.g. AppliChem A7137).3. Add 200 µl AppliCoat Plate Stabilizer per well and incubate for 15-90 minutes at 20-30°C.4a. Moist storage: Cover the plate with adhesive film and store at 2-8°C for up to 3 months.or 4b. Dry storage: Remove the AppliCoat Plate Stabilizer by aspiration. Incubate the plates at 37°C until dry. Incubation typically takes 60 to 120 minutes, depen-ding on temperature, incubator type, number of lates and the (active) air circulation in the incubator. Store the plates sealed in plastic foil or aluminium foil under dry conditions (where required with desiccant) at 2-8°C for 1 to 3 years.

products


Blocking Buffer I A7099

Solution for blocking unspecific binding sites for ELISA, EIA & Western Blots A7099,0050 50 ml A7099,0125 125 ml A7099,0500 500 ml

Composition low-molecular weight highly purified casein with NaCl and Tween®

pH value pH 7.2 ± 0.2Preservative contains 0.1 % ProClin®300Storage –20°C or at 2-8 °C Stability at –20°C: 1 year, repeated freeze / thaw cycles are possible at 2-8°C: 6 months

Instructions for use

Blocking Buffer I saturates free binding capacities on plastic consumables and other surfaces like ELISA plates and blotting membranes. Thus a reduction of unspecific binding on surfaces can be achieved.

Efficiency of blocking is significantly improved in comparison to standard blocking procedures by a special production method, which leads to casein molecules with many different molecular sizes. Blocking Buffer I can be used in ELISA, EIA, RIA, Western blotting, immuno-PCR, protein arrays as well as immunohistochemistry.

Immediately before use the buffer should be mixed thoroughly.Blocking Buffer I is ready-to-use. Repeated freezing and thawing is possible. After immobilisation of capture antibody or target protein Blocking Buffer I is applied without dilution in wells or on membranes. Incubation time has to be adopted depending on surface charac-teristics by the user. We recommend blocking over night at 4 °C, but in many cases shorter incubation is also promising.

After blocking the surface has to be washed with Washing Buffer to make it useable for the next working steps.

Blocking Buffer I Other blockers

Background reduction Extremely efficient Commonly known background reduction – background reduction even in critical assays

Usability For use in all immunoassays Some products only for use in ELISA or Western blotting, many different specialised products

Ease of application Ready-to-use For some products pre-dilution with other buffers recommended

Usability with different Usable with all common detection methods Usability depends on product, negative detection methods very good results with peroxidases, quenching with fluorescent dyes has phosphatases and fluorescent labels to be checked with some products

Effects on validation Positive effects, variations decrease, Normal effects on validation, (e.g. for new background effects are avoide, no decrease in variations shown FDA-guidance for industry) validation criteria are met easily

Storage and transportation Cooling and freezing for long-time Cooling or freezing for long-time storage, repeated freezing and storage, cooled transportation for thawing possible, but no cooled many products recommended transportation needed


1. If the plate was treated with reagents containing detergents please wash the plate 3 times in a wash buffer free of detergents (e.g. Washing Buffer TrisT-, Prod. No. A7137). If you have only used Coating Buffer (Prod. No. A7136 or A7150) aspirate Coating Buffer or empty plates by wrapping firmly onto paper cloth.

2. Add 200-300 µl Blocking Buffer II EGrade to each well. Incubate at room temperature for 1-4 hours or overnight (mostly one hour is quite enough). Duration of blocking can be minimised by shaking the plate at 600-900 rpm. Duration of blocking depends on characteristics of used surface and has to be tested individually.

3. Aspirate Blocking Buffer II EGrade or empty plates by wrapping firmly onto paper cloth. Wash 3 times in wash buffer containing a non-ionic detergent, e.g Washing Buffer TrisT+ (Prod. No. A7158).

Saturation / Blocking

mic

rotit

er p

late

s 1. If the membrane was treated with reagents containing detergent please wash the membrane 3 times in a wash buffer free of deter-gents (e.g. Washing Buffer TrisT-, Prod. No. A7137).

2. Incubate membrane in Blocking Buffer II EGrade at room tem-perature for 1-4 hours or overnight (in most cases one hour is enough). The time for blocking depends on the characteristics of the membrane used and has to be tested individually.

3. a) Wash membrane 3 times in a wash buffer containing a non-ionic detergent, e.g Washing Buffer TrisT+ (Art.-Nr. 7158).

or b) add antibody for detection and continue incubation and detection.

Blocking Buffer II EGrade A7516

Solution for blocking unspecific binding sites for ELISA, EIA, A7516,0125 125 mlWestern Blots, Protein Arrays, Immuno-PCR A7516,0500 500 ml

Composition peptide-based blocking buffer; free of serum and BSA, phosphate-freepH value pH 7.0 ± 0.2Preservative contains 0.1% ProClin® 300Storage –20°C or 2-8°C Immediately before use the buffer should be mixed thoroughlyStability at -20°C: 1 year, repeated freeze / thaw cycles are possible at 2-8 °C: 6 months

Blocking Buffer II EGrade is the most economic solution (Economical Grade) for blocking of unspecific binding sites. It is THE cost-effective alternative to the casein-based Blocking Solution I. • effective blocking • simple and economically • BSA-free


In many assays, Blocking Buffer II EGrade prevents nonspecific and unwanted binding to surfaces. Blocking Buffer II EGrade saturates free binding sites on the surfaces of e.g. microtiter plates (plastic consumables), Western blotting membranes or slides, avoiding undesired binding of analytes or detection antibodies to surfaces. This leads to significantly reduced background and improved sensitivity of the assay. Suitable for many assays, Blocking Buffer II EGrade represents an excellent alternative to BSA-based blocking solutions. Problems with interactions and cross-reactivities arising from BSA, which are present in many blocking solutions, are avoided by the use of Blocking Buffer II EGrade , as it is free of serum proteins such as BSA.

In case satisfying results cannot be obtained with the Blocking Buffer II EGrade , e.g. if your assay measures analytes in plasma, serum or tissue specimen, we strongly recommend using our Casein-based product Blocking Solution I instead (A7099).

Blocking Buffer II EGrade is a ready-to-use reagent and applied like common blockers. Use Blocking Buffer II EGrade undiluted for incubation of surfaces to saturated free binding sites in non-problematic assays.

Background and nonspecific binding can not only occur at surfaces, but also between antibodies and components of complex specimen. In this cases only an assay buffer, which is used for the immunological detection reaction, can lead to satisfactory results. Therefore we recom-mend using CrossDown Buffer (order no. A6485). CrossDown Buffer acts as a filter for binding, which doesn’t affect high affinity binding in any way, but depletes nonspecific binding and interference like matrix effects and cross reactivities.

ProClin® is a registered trade mark of Rohm and Haas Company.

mem

bran

es


products

Two – Dream Teams

Blocking Buffer III BSA A7252

Solution for blocking unspecific binding sites for ELISA, EIA, Immuno-PCR, A7252,0125 125 mlimmunohistochemistry & Western Blots A7252,0500 500 ml

Composition Standard Blocking reagent with BSA and TweenpH value pH 7.4 ± 0.2Preservative contains 0.1 % ProClin® 300Storage –20 °C or at 2-8 °C Stability at –20 °C: 1 year, repeated freeze / thaw cycles are possible at 2-8 °C: 6 months


Blocking Buffer III BSA saturates free binding capacities on surfaces of plastic consumables and other surfaces like ELISA plates and blotting membranes. Thus, a reduction of unspecific binding on surfaces can be achieved. Blocking Buffer III BSA is the standard surface blocker for many applications. If a blocker on basis of BSA (bovine serum albumin) is efficient enough for an assay, Blocking Buffer III BSA is the well-priced alternative to universally applicable and more complex blockers. Blocking Buffer III BSA can be used for ELISA, EIA, Western blotting, Immuno-PCR as well as protein arrays and immunohistochemistry.

Immediately before use the buffer should be mixed thoroughly.Blocking Buffer III BSA is ready-to-use. Repeated freezing and thawing is possible. After immobilisation of capture antibodies or target proteins Blocking Buffer III BSA is applied without dilution in wells or on membranes. The incubation time has to be adopted depending on surface characteristics by the user. We recommend blocking over night at 4 °C, but in many cases shorter incubation is also promising. After blocking, the surface has to be washed with Washing Buffer to prepare it for the next working steps.

If you find just the same background or unspecific binding in spite of correctly used Blocking Buffer III BSA, we recommend the use of Blocking Buffer I (Prod.-No. A7099). Efficiency of blocking is significantly improved with Blocking Buffer I in comparison to standard blocking procedures by the special production method of this casein containing reagent, which leads to casein molecules varying in molecular sizes. This is achieved by chemical modification from highly purified casein. Therefore, you get a maximum of safety and reproducibility. Blocking Buffer I is suited for blocking of surfaces in all immunoassays, whereas Blocking Buffer III BSA leads to sufficient results in non-problematic assays.

Background and unspecific binding can not only occur at surfaces, but also between antibodies and components of complex specimen. In this cases only an assay buffer, which is used for the immunological detection reaction, can lead to satisfactory results. Therefore, we recommend to use CrossDown Buffer (Prod.-No. A6485) for optimal results in measurements of complex and important specimen. CrossDown Buffer has an effect like a filter for binding, which doesn’t affect high affinity binding in any way, but depletes unspecific binding and interference like matrix effects and cross reactivities.



Blocking Reagent CA A3409,0010

Blocking Reagent for Hybridization Assays and Western Blots

Storage Room Temperature

The Blocking Reagent CA is used in hybridization and detection procedures using non-radioactive nucleic acid probes, and for Western blots.When immunoassays and hybridization assays, such as dot blots, Western blots, Southern blots, or Northern blots are performed, there is nonspecific binding resulting in high background. In order to reduce the nonspecific binding, Blocking Reagent CA is used to "block" unbound sites left after immobilization of the specific protein or after the hybridization with non-radioactive probe. The Blocking Reagent CA improves sensitivity and reduces background.

Note Nonfat dry milk inhibits the streptavidin-biotin interaction due to its content of biotin!

Procedure Proteins For blotting applications such as Western blots and dot blots, add 0.2 % (w/v) Blocking Reagent CA into TBST or PBST, heat to 75-80 °C in a water bath or microwave oven, and stir well until dissolved. The Blocking Reagent CA dissolves to give a milky solution. Use for blocking and for dilutions of antibodies.

Note Do not use the Blocking Reagent CA in PBST for alkaline phosphatase conjugate dilutions

Nucleic AcidsFor hybridization applications add 0.2 % (w/v) Blocking Reagent CA to Tris-Saline buffer (100 mM Tris-Cl pH 7.5, 600 mM NaCl), heat to 60 -65°C in a water bath or microwave oven, and stir well until dissolved. The Blocking Reagent CA dissolves to give a clear solution. Use for blocking after the wash steps, and before incu-bation in any enzyme-conjugate solution (e.g. Streptavidin-HRP, Streptavidin-AP).

Optimization of Time Required for Blocking with the Blocking Reagent CA

1. Cut 7 small squares of nitrocellulose or other suitable membrane.2. Label each square with a ball point pen in 10 minute increments (60, 50, 40, 30, 20, 10), and one without blocking.3. Place the first square (60) in a few ml of Blocking Reagent CA solution, and add successive squares at 10- minute intervals.4. Wash all squares in TBST, PBST or Tris-Saline buffer (100 mM Tris-Cl pH 7.5, 600 mM NaCl).5. Dilute the secondary antibody or streptavidin (HRP-conjugated) in Blocking Reagent CA solution.6. Incubate on shaker for 1 hour.7. Rinse in TBST, PBST or Tris-Saline buffer three times, 10 minutes each time.8. Detect with the Chemiluminescent Detection Kit for horseradish peroxidase (Order-No. A3417,1200).9. Evaluate background intensity in each square. Select the incubation time that gives the lowest background.

products


Coating Buffer C pH 9.6 A7150

Coating buffer for capture-antibodies and proteins on surfaces A7150,0125 125 ml

Composition Carbonate-based 10X stock solutionpH value pH 9.6 ± 0.2Preservative Buffer is delivered without any preservatives, because some preservatives can interfere with the process of coating. Thus coating buffer is safe and easy useable for many applicationsStorage –20 °C or at 2-8°C Use working solution immediately!Stability at –20°C: min. 3 months at 2 -8°C: 1 month

Instructions for useCoating Buffer C pH 9.6 is made for adsorptive immobilisation of proteins and antibodies on plastics surfaces (for example microtiter plates) or other protein binding surfaces. Applications are for example ELISA, EIA, RIA and protein arrays as well as immuno-PCR.Crystals of salt can precipitate during storage at 2-8 °C or after freezing. Therefore Coating Buffer C must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution. This leads to dissolving of salt after shaking. The stock solution is diluted 1:10 with deionized water to get the working solution. Use the working solution the same day.The proteins or antibodies for immobilisation are diluted in this working solution and used after mixing. The typical concentration range for standard ELISA is between 0.5 µg/ml and 2 µg/ml for capture antibodies. Depending on the surface as well as on proteins or antibodies the useful incubation times can differ. Consequently any user should optimise its own incubation procedure. For some proteins or antibodies Coating Buffer PBS pH 7.4 is better, for others Coating Buffer C pH 9.6 is advantages for immobilisation. The pH-value can have an influence on the steric structure of proteins or antibodies, thus having an effect on immobilisation.For an optimised procedure for a newly developed immunoassay we strongly recommend testing of both Coating Buffers in comparison.

Coating Buffer PBS pH 7.4 A7136

Coating buffer for capture-antibodies and proteins on surfaces A7136,0125 125 ml

Composition PBS-based 10x stock solutionpH value pH 7.4 ± 0.2Preservative Buffer is delivered without any preservatives, because some preservatives can interfere with the process of coating. Thus coating buffer is safe and easy useable for many applicationsStorage –20°C or at 2-8°C Stability at –20°C: 1 year at 2-8°C: min. 3 months

Instructions for useCoating Buffer PBS pH 7.4 is made for adsorptive immobilisation of proteins and antibodies on plastics surfaces (for example microtiter plates) or other protein binding surfaces. Applications are for example ELISA, EIA, RIA and protein arrays as well as immuno-PCR.Crystals of salt can precipitate during storage at 2-8 °C or after freezing. Therefore Coating Buffer PBS pH 7.4 must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution. This leads to dissolving of salt after shaking. The stock solution is diluted 1:10 with deionized water to get the working solution. Use the working solution the same day.The proteins or antibodies for immobilisation are diluted in this working solution and used after mixing. The typical concentration range for standard ELISA is 0.5 µg/ml to 2 µg/ml for capture antibodies. Depending on the surface as well as on proteins or antibodies the useful incubation times can differ. Consequently any user should optimise its own incubation procedure. For some proteins or antibodies Coating Buffer PBS pH 7.4 is better, for others Coating Buffer C pH 9.6 may have advantages for immobilisation. The pH-value can have an influence on the steric structure of proteins or antibodies, thus having an effect on immobilisation.For an optimised procedure for a newly developed immunoassay we strongly recommend testing of both Coating Buffers in comparison.


productsCrossDown Buffer A6485

Immunoassay buffer for minimisation of unspecific binding, cross-reactivities and matrix effects A6485,0050 50 ml A6485,0125 125 ml A6485,0500 500 ml

pH-Value pH 7.2 ± 0.2 Phosphat-free, ready-to-useStabilizer contains 0.1 % ProClin®300 (ProClin® is a registered trademark of Rohm and Haas)Storage –20°C or 2-8°C Stability at –20°C: 1 year, repeated freeze/thaw cycles possible at 2-8°C: 6 months


The newly developed CrossDown Buffer lowers cross reactivities, unspecific binding and matrix effects in immunoassays like ELISA, EIA, Western blotting, immuno-PCR, protein arrays, multianalyte immunoassays and immunohistochemistry – depending on the characteristics of the assay type and the used antibodies.Mix the buffer thoroughly immediately before use. CrossDown Buffer is used instead of a sample buffer or antibody dilution buffer for the immu-nological reaction. CrossDown Buffer is not suitable for blocking of surfaces. For blocking of surfaces we recommend Blocking Buffer I (Order No. A7099). CrossDown Buffer is not suited as a sample buffer for electrophoresis.

Examples of use ELISA dilution buffer for specimen and for the detection antibodies Western blotting dilution buffer for primary and secondary antibodies Immunohistochemistry dilution buffer for primary and secondary antibodies Protein arrays dilution buffer for specimen and for the detection antibodies

Dilution of the specimen Standards and specimen for ELISA and protein arrays can be diluted with CrossDown Buffer at 1:2 or higher. Standards and specimen should be treated strictly the same way.Dilution of antibodies Antibodies can be diluted with CrossDown Buffer in a user-defined manner, depending on the recommendation of the data sheet of the antibodies. This is the same for primary and secondary antibodies.Appearance of signal reduction In some cases a smooth reduc-tion of the wanted signal can be observed. CrossDown Buffer reduces low- and middle-affinity binding. That means that by the use of low- and middle-affinity antibodies or polyclonal antibodies a smooth reduction of signals can appear. Polyclonal antibodies normally contain low- and middle-affinity binding components.In the case of polyclonal antibodies a moderate increase of the concentration of the antibody can lead to the previously seen signals. Unwanted low- and middle-affinity binding will be still reduced by CrossDown Buffer. In the case of low- and middle affinity antibodies (also monoclonal antibodies) a pre-dilution of CrossDown Buffer with salt-free water can be useful to get the previously seen signal. But in this case also the unwanted bindings or cross-reactivities can partly occur again, depending on the chosen dilution with water.Although CrossDown Buffer is used as an assay buffer it is necessary to saturate surfaces like ELISA-wells or membranes with a blocking agent. We recommend the use of Blocking Buffer I (Order No. A7099). CrossDown Buffer can be used additionally as a washing buf-fer – especially in delicate or interference-sensitive assays like immu-no-PCR. Components of immunoassays – as well as of CrossDown Buffer– may quench the fluorescence of fluorescein dyes. Therefore

we recommend the use of Oyster*- (Denovo Biolabels), CyDye**- (Amersham) or Alexa***- (Molecular Probes) fluorescence dyes.We strongly recommend to test the effectiveness of CrossDown Buffer for a certain application.CrossDown in FACS analysis CrossDown buffer can replace the normally used FACS analysis assay buffer and is applied like the original assay buffer. In case that CrossDown is to “active” (i.e. reduction of the specific signal too), the most convenient way is to dilute the buffer with the original assay buffer (dilution 1 : 2 to 1 : 10). Alternatively, physiological buffers like PBS or Hepes can be used for diluting CrossDown.

*Oyster is a registered trade mark of the company Denovo Biolabels.**CyDye is a registered trade mark of the company Amersham Biosciences.***Alexa Fluor Dye is a registered trade mark of the company Molecular Probes.

Literatur [1] Miller, J.J. (2004) Clinical Laboratory International 28,

(2), 14-17

[2] Kusnezow, W., Hoheisel, J.D. (2003) J. Mol. Recognit. 16,

165-176

[3] Patton, W.F. (2000) Electrophoresis 21, 1123-1144

[4] MacBeath, G. (2002) Nat. Genet. 32, 526-532

[5] Miller, J.J., Valdes, R.Jr. (1992) J Clin Immunoassays 15,

97-107

[6] Wood, W.G. (1991) Scand. J. Clin. Lab. Invest. Suppl. 205,

105-112

[7] Kricka, L.J. (1999) Clinical Chemistry 45 (7), 942-956

[8] Span, P.N., Grebenchtchikov N., Geurts-Moespot, J., Sweep,

C.G.J. (2003) Clinical Chemistry 49 (10), 1708-1709

FAQ


FAQWhat's so special about CrossDown?

The newly developed CrossDown Buffer lowers cross reactivities, unspecific binding and matrix effects in immunoassays like ELISA, EIA, Western blotting, immuno-PCR, protein arrays, multianalyte immunoassays and immunohistochemistry. The specific, high affini-ty binding of antibody to analyte stays, while unwanted binding of the antibody is prevented.

How do I use CrossDown Buffer?

CrossDown Buffer is used instead of sample buffer or antibody dilution buffer for the immunological reaction. The sample (e.g. human serum or plasma) and the detection antibody are diluted with CrossDown Buffer, depending on the source of the interference. Assuming nonspecific binding or cross reactivity of the detection antibody, dilution with CrossDown Buffer is recommended.

Does CrossDown Buffer replace a blocking buffer for surfaces (e.g. Western blot membranes or ELISA plates)?

No! CrossDown Buffer is not suitable for blocking of surfaces, but used for dilution of the sample and/or assay antibodies instead. For blo cking

of surfaces we recommend Blocking Buffer I (Order No.

A7099).

Do I have to consider whe-ther polyclonal or monoclonal antibodies are diluted with CrossDown?No! CrossDown can be used in combi-

nation with both types of anti-body preparations. In case of polyclonal antibodies, you have to keep in mind that CrossDown only sup-ports high affinity bin-ding. Low affinity binding

is suppressed. Polyclonal antibodies are a mixture of

antibodies with different affi-nities. Therefore it may be neces-

sary to increase the concentration of a poylclonal detection antibody when used

with CrossDown Buffer. Increasing the antibody concentration increases the concentration of high

affinity antibodies as well, while the effect of low affinity antibodies will be minimized. Whether the concentration of

an antibody in an assay has to be adjusted very much depends on the quality of the antibody

Why does CrossDown Buffer assist in reducing matrix effects?

The primary cause of the so-called matrix effect is based on unwanted, low-affinity binding of matrix components (e.g. serum proteins in samples from human serum) to analytes or antibodies. The analyte may be masked by proteins or other components of the sample matrix, preventing the binding of the antibody to its target. CrossDown Buffer prevents masking and support binding of antibody and analyte.Antibodies may be masked by matrix components as well. CrossDown prevents this masking and reduces masking already present.

May I freeze-thaw the buffer several times?

The buffer can be frozen and thawed several times without a pro-blem. After thawing, the buffer has to be mixed thoroughly before application to guarantee uniform distribution of the components.

Is the buffer used diluted or undiluted?

CrossDown Buffer is a ready-to-use solution. The sample or the detection antibody can be diluted directly in CrossDown Buffer.In competitive assays and some applications in immunohisto-chemistry, it may make sense to dilute CrossDown Buffer with water or physiological buffer. The optimal conditions and ratios should be determined on a case by case basis.

Do buffer components influence color reactions?

The enzymatic activity of alkaline phosphatase or peroxidase are not negatively influenced by CrossDown Buffer. In fact, the opposite is the case. Users report an increased enzyme activity, when labeled detection antibodies were incubated with CrossDown Buffer.

Can CrossDown be applied together with flu orescent dyes, e.g. in protein chip applications?

Yes. There are positive results in protein chip applications. In some applications using a few cyan-dyes (e.g. Cy5, Cy3, oyster dyes), an increased signal was observed.


Interference effects

Background

Quality of results

Usability

Usability with different detection methods

Ease of application

Stabilisation of antibodies

Effects on validation (e.g. for new FDA-guidance for industry)

Storage and transportation

Minimisation of interference – regardless whether cross-reacti-vities, matrix effects or unspecific binding of assay components

Minimisation of background

Increase in reliability guarantees better results by avoiding interference

For use in all immunoassays

Usable with all common detection methods, very good results with peroxidases, phospha- t ases and fluorescent labels

Ready-to-use

Assay antibodies are stabilised in CrossDown Buffer, even storage of antibodies in CrossDown is possible

Positive effects, variations decrease, false results are avoided, validations can be passed successful and easy

Cooling and freezing for long-time storage, repeated freezing and thawing possible, but no cooled transportation needed

No minimisation of interference

No effects

No positive effects on reliability

Some products only for use in ELISA or Western blotting, many different specialised products

Usability depends on product, some only for use with peroxidases others only for use with phospha - tases, negative quenching with fluorescent dyes has to be checked with some products

Some products recommend pre-dilutions with other buffers

No effects on stability of assay antibodies

No positive effects on validation, interference like matrix effects or cross-reactivities lead to high variations or false results

Cooling or freezing for long-time storage, most products recommend cooled transportation

Minimisation only of interference derived from HAMAs – (Human Anti Mouse Antibodies) – All other inter-ference effects lead to wrong results!

Effects only if background comes from HAMAs

Increase in reliability only when specimen / samples include HAMAs

For use only for human specimen/ samples

Usability depends on product, some only for use with peroxidases others only for use with phosphatases, negative quenching with fluorescent dyes has to be checked with some products

Some products recommend pre-dilutions with other buffers

No effects on stability of assay antibodies

Positive effects only if HAMAs are inside the samples. Then false results can be avoided. No effect on matrix effects and other cross-reactivities

Cooling or freezing for most products necessary, cooled transportation needed

CrossDown Buffer Antibody Diluent HAMA-Blocker

Whyusing CrossDown Buffer??


CrossDown Peroxidase-Stabilizer A8625

l Assay-diluent for one-step incubation assays A8625,0050 50 mll peroxidase conjugate stabilizer A8625,0125 125 mll substitute for HAMA blocker A8625,0500 500 ml

pH value pH 7.2 ± 0.2Preservative contains 0.1 % ProClin®300 (ProClin® is a registered trademark of Rohm and Haas)Storage 2-8°C. Do NOT freeze. May form a precipitate.Stability 24 months

DescriptionIn one-step incubation assays the specimen is incubated directly with the detector-conjugate saving additional washing steps. This is very convenient and enables for very fast assay protocols in comparison to sequential procedures. Unfortunately this significantly increases the risk of interference, matrix effects and problems due to HAMAs (human anti-mouse antibodies) and rheumatoid factors. For this reason reliability suffers in favour of user-friendliness. CrossDown Peroxidase-Stabilizer solves this problem. CrossDown Peroxidase-Stabilizer enables assays with the highest reliability and robustness by minimising interference. At the same time CrossDown Peroxidase-Stabilizer is a premium stabi-lizer for peroxidase conjugates. Therefore conjugates can be stored directly in very low ready-to-use concentrations. Thus convenience and reliability are no longer incompatible.

Instructions for useCrossDown Peroxidase-Stabilizer is a dilution buffer for long-term storage of peroxidase-conjugates. This can be antibody-peroxidase-con-jugates as well as Neutravidin- or Streptavidin-peroxidase-conjugates. Conjugates can be diluted directly in CrossDown Peroxidase-Stabilizer to the final concentrations. Typical concentrations of conjugates are ranging from 40 to 500 ng/ml.Immediately before use the buffer should be mixed thoroughly.CrossDown Peroxidase-Stabilizer is a combination of CrossDown Buffer and a new stabilizer for stabilizing antibodies and peroxidase. Therefore you get long-term stabilization as well as the CrossDown Buffer effect in one solution.The CrossDown Buffer effect:CrossDown Buffer was specially developed to minimise cross reactivities, nonspecific binding and matrix effects in immunoassays. Interference derived from HAMA (human anti-mouse antibodies) rheumatoid factors or other endogenous interfering factors can be reduced significantly.Observation of signal reduction:In some cases a slight reduction of the wanted signal can be observed. CrossDown Buffer reduces low and medium-affinity binding. That means that by using low- and medium-affinity detector-antibodies or polyclonal detector-antibodies a smooth reduction of signals can appear. Polyclonal antibodies normally contain low- and medium-affinity binding components. In the case of polyclonal antibodies a moderate increa-se of the concentration of the antibody can lead to the previously seen signals.

HRP – long-term stabilization of peroxidase:CrossDown Peroxidase-Stabilizer contains stabilizing compon-ents for antibodies and for peroxidase. This makes CrossDown Peroxidase-Stabilizer the ideal solution, if you want to store peroxidase-conjugates for a long period in a ready-to-use solution. Dilute the conjugates directly in CrossDown Peroxidase-Stabilizer.Stabilization of a HRP-antibody conjugate by a competitor's enzyme stabilizer product was compared to stabilization by CrossDown Peroxidase-Stabilizer (see fig. 1). CrossDown Peroxidase-Stabilizer provides the best long-term stabilization of the HRP-antibody con-jugate. Testing CrossDown Peroxidase-Stabilizer with any new assay is recommended.Stability data of one peroxidase-conjugate cannot be transferred to other conjugates. Therefore every conjugate has to be tested for its shelf life in CrossDown Peroxidase-Stabilizer. If CrossDown Peroxidase-Stabilizer is used for immunodiagnostic kits, the shelf life has to be tested according to the current European directive for invitro-diagnostics or according to corresponding rules, which apply.

Figure 1: ShelflifeofHRP-antibodyconjugateboundtotheanalyteismeasuredasperoxidasesignalinanELISAandgivenas%valueoffreshlydilutedHRP-antibodyconjugate.Detector-conjugateconcentra-tionwas40ng/ml.

products


Peroxidase-Stabilizer A8647

dilution buffer and storage solution for peroxidase-conjugates for an outstanding shelf-life of the conjugates A8647,0050 50 mll protection and stability of your HRP-conjugate for years A8647,0125 125 mll Peroxidase-Stabilizer maintains enzyme activity A8647,0500 500 mll Peroxidase-Stabilizer maintains functionality of HRP-conjugatel conjugate may be stored at very low final concentration – no pre dilution steps

pH value pH 7.2 ± 0.2Preservative contains 0.1 % ProClin®300Storage 2-8°C. Do NOT freeze. May form a precipitate.Stability 24 months

DescriptionStabilization of all active components is very critical to get reliable results with immunoassays. When assays are stored for long-term before use, it is especially important to ensure adequate stability. Peroxidase-Stabilizer works very well as a stabilizer. Additionally it is a very good assay buffer. This enables any user to store HRP conjugates directly in ready-to-use concentrations, even when these concentrations are very low. This saves pre-dilution steps before doing an assay. Peroxidase-Stabilizer maintains the activity of conjugates of antibodies and of Neutravidin conjugated to HRP. Therefore it can be used in any assay, in which HRP is the detector enzyme. Peroxidase-Stabilizer decreases the variability often caused by changing shipping and storage conditions, thereby producing more consistent results with your diagnostic test kit. Peroxidase-Stabilizer can be easily incorporated into most assay protocols by substituting it for your conjugate diluent.

Instructions for useConjugates can be diluted directly in Peroxidase-Stabilizer in its final concentration. Typical concentrations of conjugates are ranging from 40 to 500 ng/ml. Peroxidase-Stabilizer can be used directly as assay buffer in immunoassays. Peroxidase-Stabilizer is designed for trouble-free assays without interference. If you have background issues or observe false-positives – for example derived from cross reactivities, matrix effects or HAMA’s – we recommend using CrossDown-Buffer or CrossDown Peroxidase-Stabilizer rather than Peroxidase-Stabilizer.Suitability of Peroxidase-Stabilizer for a specific assay has to be tested by the user.Immediately before use the buffer should be mixed thoroughly.Peroxidase-Stabilizer – long-term stabilization of peroxidase:Peroxidase-Stabilizer contains stabilizing components for antibodies and for peroxidase. This makes Peroxidase-Stabilizer the ideal solution, if you want to store peroxidase-conjugates for a long period in a ready-to-use solution. Dilute the conjugates directly in Peroxidase-Stabilizer.

Stability testStability „stress“ tests were performed with Peroxidase-Stabilizer (PS) using a monoclonal antibody conjugate (see fig. 1). HRP-conjugate of the antibody was stabilized either by a so far leading enzyme stabilizer product or by Peroxidase-Stabilizer. Stress tests were performed at 45°C over a period of up to 143 days. Percent retained activity was determined by comparing the activity of the HRP-antibody conjugate bound to the analyte. After 143 days incubation at 45°C with Peroxidase-Stabilizer the monoclonal antibody conjugate retained around 80 % enzymatic HRP activity. This shows a shelf life at 4°C with approximately 80% enzymatic activity of more than 6 years by conservative correlation according to Arrhenius. This is a very good stability and therefore Peroxidase-Stabilizer gives an optimum product safety for HRP containing immunoassays!Testing Peroxidase-Stabilizer with any new assay is recommended.Stability data of a peroxidase-conjugate cannot be transferred to another conjugate. Therefore every conjugate has to be tested for its shelf life in Peroxidase-Stabilizer. If Peroxidase-Stabilizer is used for immunodiagnostic kits, the shelf life has to be tested according to the current European directive for invitro-diagnostics or according to corresponding rules, which apply.

Figure 1: ShelflifeofHRP-antibodyconjugateboundtotheanalyteismeasuredasperoxidasesignalinanELISAandgivenas%valueoffreshlydilutedHRP-antibodyconjugate.Detector-conjugateconcentra-tionwas40ng/ml.

products


Sample Buffer T- A7101

Sample buffer and dilution buffer for antibodies for use in immunoassays and immunohistochemistry A7101,0050 50 ml A7101,0125 125 ml A7101,0500 500 ml

Composition detergent-free, phosphate-freepH value pH 7.2 ± 0.2Preservative contains 0.1 % ProClin®300Storage –20 °C or at 2-8 °C; repeated freezing and thawing cycles possible Stability at –20 °C: 1 year ; at 2-8 °C: 6 months


Sample Buffer T- is a dilution buffer for specimen and antibodies für direct use in immunoassays. Antibodies, coupled to alkaline phosphatase or peroxidase, can be diluted and used in Sample Buffer T- without problems, because Sample Buffer T- is free of phosphate.Sample Buffer T- without detergents contains neither Tween® nor other detergents. Thus it is for use especially in immunohistochemistry, where detergents sometimes can make problems in detection.

Immediately before use the buffer should be mixed thoroughly. Sample Buffer T- is ready-to-use. Repeated freezing and thawing is possible. Primary and secondary antibodies can be diluted directly in Sample Buffer T-.

In case you observe increased background or unwanted matrix effects in the presence of Sample Buffer T-, we recommend the use of CrossDown Buffer (Artikel-Nr. A6485) instead of Sample Buffer T-.

Sample Buffer T+ A7134

Sample buffer and dilution buffer for antibodies for use in immunoassays A7134,0050 50 ml A7134,0125 125 ml A7134,0500 500 ml

Composition contains Tween® pH value pH 7.2 ± 0.2Preservative contains 0.1 % ProClin®300Storage –20 °C or at 2-8 °C; repeated freezing and thawing cycles possible Stability at –20 °C: 1 year ; at 2-8 °C: 6 months


Sample Buffer T+ is a dilution buffer for specimen and antibodies für direct use in immunoassays. Antibodies, coupled to alkaline phospha-tase or peroxidase, can be diluted and used in Sample Buffer T+ without problems, because Sample Buffer T+ is free of phosphate.

Sample Buffer T+ can be used for ELISA, EIA or Western blotting as well as for immuno-PCR or protein arrays. Sample Buffer T+ is not for use as an electrophoresis buffer.

Immediately before use the buffer should be mixed thoroughly.Sample Buffer T+ is ready-to-use. Repeated freezing and thawing is possible. Specimen with the analyte – as well as the detecttion antibody – is diluted in Sample Buffer T+ and then used in the assay. It is necessary to treat standards and specimen in the same way!

In case you observe increased background or unwanted matrix effects in the presence of Sample Buffer T+, we recommend the use of CrossDown Buffer (Artikel-Nr. A6485) instead of Sample Buffer T+.

Stripping Buffer I A7140

Stripping buffer for Western blots for multiple reprobing A7140,0050 50 ml A7140,0125 125 ml Composition doesn't contain β-mercaptoethanol and DTTpH value pH 2.8 ± 0.2Preservative contains 0.1 % ProClin®300Storage –20 °C or at 2-8 °C Stability at –20 °C: 1 year at 2-8 °C: 6 months


Ready-to-use

Stripping Buffer I removes reaction solution, primary and secondary antibodies from Western blotting membranes. After stripping the mem-brane can be used for a second detection (reprobing) with the same or other antibodies.

The membrane is incubated in a vessel with Stripping Buffer I for removing the antibodies. For this purpose gently shake the membrane in Stripping Buffer I for 30-60 minutes at room temperature.

After stripping, the membrane has to be washed with Washing Buffer and can be used for a second detection. If you detect with alkaline phosphatase, the wash buffer should not contain phosphates.

Important: The designated incubation conditions are standard values, which have to be adopted by the user. Incubation times are depending on characteristics of the used antibodies.

Washing Buffer TrisT- (10X) A7137

Washing buffer for use in ELISA, EIA, Western blotting and immunohistochemistry A7137,0500 500 ml Composition Tris-based 10X buffer with 350 mM NaCl; contains no detergentspH value pH 7.2 ± 0.2Preservative contains 0.1 % ProClin®300Storage –20 °C oder bei 2-8°C Use working solution immediately!Stability at –20 °C: 1 year at 2-8 °C: 6 months


This Washing Buffer TrisT- is made especially for immunohistochemistry. It should be used, whenever detergents can make problems in detection.

Crystals of salt can precipitate during storage at 2-8 °C or after freezing. Therefore Washing Buffer TrisT- must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution. This leads to dissolving of salt after shaking. The stock solution is diluted 1:10 with deionized water to get the working solution. Use the working solution the same day.


products

Washing Buffer TrisT+ (10X) A7158

Washing buffer for use in ELISA, EIA and Western blotting A7158,0500 500 ml Composition Tris-based 10X buffer; contains Tween® and 350 mM NaClpH value pH 7.2 ± 0.2Preservative contains 0.1 % ProClin®300Storage bei –20 °C or at 2-8°C Use working solution immediately!Stability at –20 °C: 1 year at 2-8 °C: 6 months


Washing Buffer TrisT+ is used for many immunoassays. Washing steps are needed to remove unbound and excessive components, which are able to interfere with the assay. Application fields are ELISA, EIA, RIA, Western blotting as well as immuno-PCR, protein arrays and multianalyte immunoassays. It is applicable in automatic plate washers depending on salt tolerance of the washer.

Caution Make sure that you have checked the specifications and instructions of your washer!

Crystals of salt can precipitate during storage at 2-8 °C or after freezing. Therefore Washing Buffer TrisT+ must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution. This leads to dissolving of salt after shaking. The stock solution is diluted 1:10 with deionized water to get the working solution. Use the working solution the same day.

Especially for use in immunohistochemistry we offer Washing Buffer TrisT- without Tween or other detergents (Product No. A7137).

35


Blocking Reagents Prod. No.

Albumin acetylated A0845Albumin Fraction V (pH 7.0) Blotting grade A6588Denhardt's - Solution (50X) BioChemica A2248Denhardt's powder mixture (for 50X stock solution) A3792Dextran sulfate 500 sodium salt BioChemica A2250Dextran sulfate 500 sodium salt Molecular biology grade A4970Gelatin powdered pure Ph. Eur., NF A1693Heparin sodium salt A3004Nonfat dried milk powder A0830Polyvinylpyrrolidone (K90) Molecular biology grade A2260Salmon sperm DNA sodium salt A2160Salmon sperm DNA sodium salt (sonified) A2159

Transfer membranes Prod. No.

Reprobe Nitrocellulose supported 0.22 µm Transfer membrane A5237Reprobe Nitrocellulose supported 0.45 µm Transfer membrane A5242Pure Nitrocellulose unsupported 0.22 µm Transfer membrane A5250Pure Nitrocellulose unsupported 0.45 µm Transfer membrane A5239Pure Nylon Neutral Transfer membrane 0.22 µm (30 cm x 3 m) A4399Pure Nylon Neutral Transfer membrane 0.45 µm A5248Reprobe Nylon Positively charged Transfer membrane 0.45 µm (30 cm x 3 m) A5255PVDF-Star Transfer membrane 0.45 µm A5243

Brochure Transfer Membranes

Other Related Products Prod. No.

Chemiluminescence Detection Kit for Horseradish Peroxidase A3417Peroxidase fom horseradish EIA and Immunology Grade I A3615Peroxidase fom horseradish EIA and Immunology Grade II A3771Sodium azide pure A1430Streptavidin ultrapure A1495Thimerosal BioChemica A1278

Further readingArnold M. Raem & Peter Rauch (Hrsg.) Immunoassays 2007 Elsevier Spektrum Akademischer Verlag (ISBN 3-8274-1636-1) german language

David Wild (Ed.): The Immunoassay Handbook. 3rd Edition. Elsevier Science Publishing Company, Amsterdam, Boston, Oxford 2005, ISBN 0-08-044526-8

Werner Luttmann, Kai Bratke, Michael Küpper, Daniel Myrtek: Der Experimentator: Immunologie. 2. Auflage. Spektrum Akademischer Verlag, Heidelberg 2006, (ISBN 3-8274-1730-9) german language

Related products

infotaining

Transfer Membranes

AppliChem supplies a range of transfer membranes designed and tested speci-fically for RNA, DNA and protein analysis. AppliChem also provides our customers with tried and proven protocols developed to obtain consistent repro-ducible and dependable results when used with AppliChem membranes. 22 protocols and all types of membranes – all from one source.

Gel Electrophoresis Size MarkerReady-to-use DNA size marker and protein size marker for gel electro-phoresis are available from many sources. Besides these standard products, AppliChem offers the full range of lyophilized DNA markers. The advantage of lyophilized marker is their outstanding stability. The long shelf life of more than 5 years is achieved by deproteinization and subsequent lyophilization. Read more about the usage of our markers in our new brochure.

Get your free copy – www.applichem.com

Biological Buffers Many biochemical processes are markedly impaired by even small changes in the concentration of free H+ ions. It is therefore usually necessary to stabilise the H+ concentration in vitro by adding a suitable buffer to the medium, without, however, affecting the functioning of the system under investi-gation.Biological buffers are an essential part of each experiment. Several aspects have to be taken into account, when planning an experiment. AppliChem‘s brochure “Biological Buffers” imparts basic knowledge on the criteria for selecting the right buffer and requirements of buffers.

DetergentsDetergents are more than just air bubbles. As diverse as the research objects and techniques are as diverse are the detergents available. The proper-ties of e.g. SDS and octylglucoside are so different that you hardly may exchange them for the identical experiment. Why do you apply SDS in gel electro-phoresis but not dodecylmaltoside? Because your colleagues did it all the time before? AppliChem‘s brochure „Detergents“ will help you selecting the best detergent for your assay.

Von Wissenschaftlern für Wissbegierige

in der Chemie, der Biotechnologie und Pharmaforschung 5/08

Think!

Forschst Du noch

oder denkst Du schon?

Faszinierende EinblickeHirnwelt Medienwelt

Flimmernde Realität

UnterweltErschreckende Dimensionen

Medienwelt

The contamination of cells with mycoplasma is a very com-mon problem, even though it often goes unno-ticed since no cloudiness appears in the cell culture. Mycoplasma are small and may pass the filtration units applied for preparing cell culture media. They do influence the growth, morphology and survival of the host cells, resulting in a strong influence on the test system and the results obtained. AppliChem offers a PCR-based test kit to test for infection, antibiotics for the treatment of infected cells, and cleansing reagents for CO2-incubators and water-baths to prevent infection.

Safety First: Banish Mycoplasma

Get AppliChem‘s phantastic customer magazine free of charge: available in English and [email protected]

There is another top address in Darmstadt:AppliChem GmbH Ottoweg 4 D - 64291 Darmstadt Phone +49 6151 9357-0 Fax +49 6151 9357-11

eMail [email protected] internet www.applichem.com

4t M

atth

es +

Tra

ut ·

Darm

stadt Immunoassay

Buffer

A71,E

08/2010

Download - Immunoassay Buffer

Top Related