ENZYME IMMUNOASS

AYCantuba, Abby Camille

Pasia, Kristine May

Reserone, Ysabel

Ganotisi, Rowena

MD3Y2-1

IMS 324

INTRODUCTION Enzymes are naturally occurring

molecules that catalyze certain biochemical reactions.

They react with suitable substrates to produce breakdown products that may be chromogenic, fluorogenic, or luminescent.

Sensitivity can be achieved without disposal problems or the health hazards of radiation.

Because one molecule of enzyme can generate many molecules of product, little reagent is necessary to produce high sensitivity.

Assays based on the use of enzymes can be found in such diverse settings as clinical laboratories, doctors’ offices, and at-home testing.

Typical enzymes that have been used as labels in colorimetric reactions include horseradish peroxidase, glucose-6-phosphate dehydrogenase, alkaline phosphatase, and -D-galactosidase.1,4 Alkaline phosphatase and horseradish peroxidase are most often used in such assays.

EIAs can be applied to all Ag–Ab systems, including those involving serum protein, hormones, drugs, and other antigens and the antibodies directed against pathogens.

Quantitative immunoassays using enzymes as labels were developed as alternatives to radioisotopes.

The most widely used are:enzyme-linked immunosorbent

assay (ELISA)enzyme-multiplied immunoassay

technique (EMIT), which is a registered trade name of SYVA Co.

ENZYME LABELS Enzymes used as labels for

immunoassay are typically chosen according to the number of substrate molecules converted per molecule of enzyme, ease and speed of detection, and stability. In addition, availability and cost of enzyme and substrate play a role in the choice of a particular enzyme as reagent.

To be suitable as a label an enzyme must meet several criteria:

ADVANTAGES OF ENZYME IMMUNOASSAY Labels are cheap and plentiful. Labels have a long shelf life Easily adapted to automation Reaction measured using inexpensive

equipment Very sensitive No health hazards associated with

reagents Can be used for qualitative OR

quantitative procedures.

DISADVANTAGES OF ENZYME IMMUNOASSAY Some samples may have natural

inhibitors. Size of enzyme label limiting factor in

designing some assays. Nonspecific protein binding may

occur. Enzyme reactions very sensitive to

temperature.

CLASSIFICATION Enzyme assays are classified as either

heterogeneous or homogeneous on the basis of whether a separation step is necessary.

Heterogeneous enzyme immunoassays require a step to physically separate free from bound analyte. In homogeneous immunoassays, on the other hand, no separation step is necessary, because enzyme activity diminishes when binding of antibody and antigen occurs.

HETEROGENEOUS ENZYME

IMMUNOASSAY

The assay principle of heterogeneous EIAs is similar to that of RIAs, except that enzyme activity, not radioactivity, is measured

EIAs require a secondary process to obtain signals through the catalytic reaction of enzymes

Microtiter plate wells, plastic beads, plastic tubes, magnetic particles, and latex with filters can be used as the solid phase for the separation of bound and free conjugates.

The development of substrates to be cleaved by enzymes was marked by the introduction of colorimetric and fluorometric substrates, and later chemiluminescent substrates, which increased the signal sensitivity.

Enzyme Used:

horseradish peroxidase alkaline phosphatase β-galactosidase glucose oxidase urease catalase

There are two versions of the competitive format:

One – step format:

Two – step format:

Competitive EIA

Competitive EIA In a competitive format, unlabeled

analyte (usually the antigen) in the test sample is measured by its ability to compete with the labeled antigen in the immunoassay

In a competitive immunoassay, less label measured in the assay means more of the unlabeled (test sample) antigen is present

Non-Competitive EIAPrinciple:

Non-Competitive EIA often referred to as indirect enzyme-linked

immunosorbent assays (ELISA)

enzyme-labeled reagent does not participate in the initial antigen–antibody binding reaction

used to measure antibody production to infectious agents that are difficult to isolate in the laboratory and has been used for autoantibody testing

one of the most frequently used immunoassays in the clinical laboratory due to its sensitivity, specificity, simplicity, and low cost

either antigen or antibody may be bound to solid phase

a variety of solid-phase supports are used, including microtiter plates, nitrocellulose membranes, and magnetic latex beads

Viral infections especially are more easily diagnosed by this method than by other types of testing

preferred screening method for detecting antibody to HIV, hepatitis A, and hepatitis C

ELISA-based tests are also used to identify Epstein-Barr–specific antibodies produced in infectious mononucleosis

Sandwich ImmunoassayPrinciple:

Sandwich Immunoassay

if antibody is bound to the solid phase, these assays are often called sandwich immunoassays, or capture assays

best suited to antigens that have multiple determinants, such as antibodies, polypeptide hormones, proteins, tumor markers, and microorganisms, especially viruses

use of monoclonal antibodies has made this a very sensitive test system

recently developed ELISAs have made it much easier to detect parasites such as Giardia lamblia and Cryptosporidium in the stool

Antigen-detection tests have also proved useful for identifying fungi such as Aspergillus, Candida, and Cryptococcus

Sandwich Immunoassay the presence of IgM can be specifically determined,

thus indicating an acute infection

measurement of IgE, including allergen-specific IgE, which appears in minute quantities in serum, can also be accomplished with this system

in sandwich assays, capture antibody on solid phase must have both a high affinity and a high specificity for this test system to be effective

sandwich assays are also subject to the hook effect, an unexpected fall in the amount of measured analyte when an extremely high concentration is present

COLORIMETRIC ENZYME

IMMUNOASSAY

In this assay, enzyme reaction is performed by using chromogenic substrates to develop a color by prime catalytic reaction

A spectrophotometer is used to measure the optical density of the resulting chromogen

When the EIA reaction is performed on nitrocellulose membrane (Western blotting method) or other membranes, substrates that generate insoluble dyes are utilized

An hCG pregnancy test with an immunoassay format that is sold as an over-the-counter test commonly uses benzidine derivatives to react with peroxidase, or indoxyl phosphate derivatives to react with alkaline phosphatase, to generate insoluble dyes

The measurement of optical density is limited to a range of 0–2.0

the determination of optical density for analytes that require determination of a wide dynamic range can be problematic, even when an excess amount of conjugate and a solid phase with sufficient capturing capacity are used with a sandwich-type assay

Ishikawa (1989) developed an ultrasensitive EIA capable of detecting 3 fmol of specific IgG antibody

to achieve this level of sensitivity, several tedious steps are required, such as immunocomplex transfer from a first solid phase to another solid phase to reduce background signals

Enzyme Used:

horseradish peroxidase catalyzing ABTS (diammonium salt of 2’2-

azinodi[{3-ethyl-benzothione-6-sulfonate}] w/ H2O2, to form a green color

alkaline phosphatase specific to the p-nitrophenylphosphate, to form a yellow color

FLUORESCENT ENZYME

IMMUNOASSAY

Fluorescent EIAs are identical to other EIAs, except that they use fluorescent substrates.

In the fluorescent EIA, a fluorophore is generated by an enzyme reaction. Following excitation of the fluorophore at its optimal light excitation wavelength, light at a characteristic wavelength is emitted. Instruments such as a fluorometer require both a supplier of the excitation light source and a photomultiplier tube as a detector of the emission fluorescence.

Thus, close attention should be paid to the selection of substrates for EIAs to avoid interfering factors.

Compared with colorimetric EIAs, fluorescent assays generate a signal intensity that is at least one order of magnitude greater.

CHEMILUMINESCENT ENZYME

IMMUNOASSAY

Chemiluminescent enzyme immunoassays (CL-EIAs) use chemiluminescent substrates that react with various enzymes employed as labels.

The chemiluminescent enzymatic reaction generates light, similar to bioluminescence, which involves the use of natural substrates such as luciferin–adenosine triphosphate.

The enzymatic oxidation reaction of luminol analogs has long been used for CL-EIA. Use of peroxidase with H2O2 is a common method that is interchangeable with an alternative coupling enzyme producing H2O2, such as glucose oxidase or uricase.

A chemiluminescent signal with maximum wavelength of 477 nm can be detected within a couple of minutes to a few hours, depending on substrate concentration.

The alkaline phosphatase with AMPPD assay system has a sensitivity of less than 10−20 mol

CL-EIA can be performed on a fully automated instrument. This novel substrate made it possible to develop an extremely sensitive chemiluminescent enzyme immunoassay system.

HOMOGENEOUS ENZYME

IMMUNOASSAYS

Homogeneous enzyme immunoassays have played a major role in the development of simple and easy to use diagnostic tests for clinical laboratory instrumentation

Enzymes and their cofactors are advantageous labels in homogeneous EIAs.

enzyme activity can be modulated easily by changing factors in the microenvironment of the Ag–Ab reaction.

Homogeneous EIAs are generally less sensitive than their heterogeneous counterparts.

Conventional heterogeneous EIAs have equal sensitivity to RIAs in many applications while homogeneous EIAs remain one or two orders of magnitude less sensitive than RIAs.

Homogeneous EIAs may require complex immunochemical reagents.

the assay systems are rapid and simple and are adaptable to conventional instruments.

HOMOGENEOUS EIASCAN BE CLASSIFIED AS :

COMPETITIVE assays usually consist of enzyme-labeled

antigens.

The antigen (analyte) may be conjugated to the substrate or to a prosthetic group of the enzyme in other assay formats.

NON-COMPETITIVE use a conjugate of antibody labeled with enzyme

CLASSIFICATION & CHARACTERISTICS OF HOMOGENEOUS EIAS

In each of these assays (Table 44-7) the Ag–Ab interaction modulates the

activity of the enzyme or enzyme label in the presence of the substrate.

Modulation of the enzyme activity reflects the degree of the immunochemical reaction.

EIA methods have been developed with comparable sensitivity to those of radioimmunoassay (RIA).

EIAs have become established in many clinical laboratories and are generally being employed for:

1) homogeneous assay of low molecular weight compounds such as drugs and antibiotics present in biological samples at relatively high concentrations,

2) qualitative and semiquantitative assays for viral, bacterial, fungal, and parasitic antigens and antibodies,

3) tissue immunohistochemical localization of antigens and antibodies, and

4) alternatives to RIA in smaller laboratories.

 ENZYME-MULTIPLIED IMMUNOASSAY

TECHNIQUE

EMIT, the first homogeneous EIA, was developed by Rubenstein (1972).

In this assay , the conjugation of enzymes to haptens does not disrupt enzyme activity; however, the binding of hapten-specific antibodies to haptens results in inhibition of enzyme activity. Free haptens in the standard or sample relieve this inhibition by competing for antibodies.

in the presence of antibodies, enzyme activity is proportional to the concentration of free haptens.

As a general rule, the antibody inhibits the enzyme by inducing or preventing conformational changes necessary for enzyme activity (Rowley, 1975).

The exception to this inhibition mechanism is the EMIT thyroxine assay, which uses malate dehydrogenase.

the thyroxine-malate dehydrogenase conjugate in this assay is enzymatically inactive, but it becomes activated when it is bound by thyroxine antibody (Ullman, 1979).

Conjugated thyroxine inhibits the enzyme by binding to the active site, thus increasing the “apparent” Km of the substrate.

The antibody reactivates the enzyme by “pulling” the thyroxine out of the active site.

THE EMIT SYSTEM

In the EMIT assay system, shown in (figure 44.15) malate dehydrogenase and glucose-6-phosphate dehydrogenase have been found to be most useful because they are less likely to be affected by serum constituents.

These assays generally measure drugs in a concentration of milligrams per liter and also measures drug metabolites (drug-derived molecules) in biological fluids, such as blood and urine.

The following drugs are detected by EMIT: cocaine and metabolites, cannabinoides, opiates, amphetamines, phentanyl, methadone, barbiturates, benzodiazepines, phenylciclidine, and propoxyphene.

the digoxin assay has a much lower limit of sensitivity in the range of 0.8–2 μg/L.

SUBSTRATE-LABELED

FLUORESCENT IMMUNOASSAY

substrate-labeled fluorescent immunoassay (SLFIA) uses a characteristic of fluorogenic substrate, umbelliferyl β-galactoside, attached to the antigen (analyte) as a conjugate.

Umbelliferone is the fluorescent product produced when the substrate is cleaved with the enzyme β-galactosidase, which cannot cleave the substrate–antigen complex when it is reacted with the specific antibody.

SUBSTRATE-LABELED FLUORESCENT IMMUNOASSAY

The free antigen (analyte) in the specimen solution competes with the antigen conjugated with the substrate to form the immunocomplex (Fig. 44-16)

The antigen concentration in the sample is proportional to the fluorescent intensity of the cleaved fluorescent product.

USES OF SLFIA

can be used to assay drugs and haptens, as well as protein ligands such as IgG and IgM.

A homogeneous substrate-labeled fluorescent immunoassay has been applied to the measurement of phenytoin concentrations in human serum.

A coupled fluorogenic enzyme substrate, galactosyl-umbelliferone, covalently to a derivative of phenytoin.

DISADVANTAGE OF SLFIA

disadvantage of this method is that the amplification properties of the enzyme are not utilized, and thus the assay system has limited sensitivity in the range of 10−9–10−10 molar concentration of the analyte.

APOENZYME REACTIVATION IMMUNOASSAY

a homogeneous assay developed by Morris (1981) using the prosthetic group consisting of flavin adenine dinucleotide (FAD)-conjugated antigen (analyte) and glucose oxidase apoenzyme.

ARIS has been used to assay for theophylline and IgG (Morris, 1981, 1985). This FAD-labeled conjugate assay is readily adapted to the measurement of high molecular weight proteins (e.g., thyroid binding globulin) (Schroeder, 1985), as well as other haptens such as phenytoin and hormones.

the antigen (analyte) and a constant amount of analyte–FAD conjugate compete for a limited amount of specific antibody.

At equilibrium, the level of free conjugate is proportional to the amount of antigen (analyte) in the specimen.

The apoenzyme combines with the free but not with the antibody-bound form of conjugate to reactivate glucose oxidase activity in proportion to the amount of free conjugate in the mixture.

Active enzyme is generated in the procedure, and an amplification mechanism is built into this assay.

ENZYME INHIBITORY HOMOGENOUS IMMUNOASSAY

developed by Ashihara (1988), consists of antibody conjugated with enzyme and insoluble substrate. This assay is most suitable for the determination of large antigens (analytes).

can be made to be homogeneous because the immunocomplex of conjugate with large antigen blocks the enzyme reaction with the solid substrate. α-Amylase has been used as a labelled enzyme for the determination of ferritin and AFP.

EIHIA based on a noncompetitive binding assay requires less incubation time to achieve a sensitive detection level.

EIHIA has also been applied to a simple dryfilm format.

The dry film consists of three major layers, each containing: developing zone for immunologic and enzymatic

reaction a barrier zone color-developing zone.

An inhibitor specific to human amylase present in serum is used to prevent serum background.

The assay for serum C-reactive protein is completed in only 6 minutes by simply placing the specimen on the slide using dry chemistry instrumentation.

the sensitivity of the system remains inadequate for application to analytes such as tumor markers.

CLONED ENZYME DONOR

IMMUNOASSAY

first achieved through the application of recombinant DNA technology to homogeneous immunoassays by Henderson (1986).

Microgenics Corp. (Concord, Calif.) was able to engineer β-galactosidase protein into a large polypeptide (an enzyme acceptor [EA]) and a small polypeptide (an enzyme donor [ED]).

EAs and EDs assemble to form enzymatically active tetramers.

In the assay shown in the diagram, a hapten antigen (analyte) is attached to an ED, and an analyte-specific antibody is used to inhibit spontaneous assembly of the active enzyme.

The antigens (analytes) in patient serum compete with the analytes in the analyte–ED conjugate for antibody, modulating the amount of active β-galactosidase formed.

The signal generated by enzyme substrates is directly proportional to the analyte concentration in the patient serum.

The test for digoxin is a colorimetric assay that requires no serum pretreatment or predilution. The assay system is suitable for use with automated chemistry analyzers.

MEMBRANE-BASED

CASSETTE ASSAYS

Membrane-based cassette assays are a relatively new type of enzyme immunoassay.

They are rapid, easy to perform, and give reproducible results.

designed primarily for point-of-care or home testing

many of these have been modified for increased sensitivity and can be made semiquantitative for use in a clinical laboratory.

Typically these are designed as single-use, disposable assays in a plastic cartridge. The membrane is usually nitrocellulose, which is able to easily immobilize proteins and nucleic acids.

The rapid flow through the membrane and its large surface area enhance the speed and sensitivity of ELISA reactions.

Either antigen or antibody can be coupled to the membrane, and the reaction is read by looking for the presence of a colored reaction product.

Some test devices require the separate addition of patient sample, wash reagent, labeled antigen or antibody, and the substrate.

Another type of rapid assay, called immunochromatography.

The analyte is applied at one end of the strip and migrates toward the distal end, where there is an absorbent pad to maintain a constant capillary flow rate.

The labeling and detection zones are set between the two ends.

As the sample is loaded, it reconstitutes the labeled antigen or antibody, and the two form a complex that migrates toward the detection zone.

Excess labeled immunoreactant migrates to the absorbent pad

This type of test device has been used to identify microorganisms such as Streptococcus pyogenes and Streptococcus agalactiae and has been used to test for pregnancy, for troponin in a heart attack, and for hepatitis B surface antigen, to name just a few examples.

Test results are most often qualitative rather than quantitative.

ENZYME-LINKED IMMUNOSORBENT

ASSAY (ELISA)

ENZYME-LINKED IMMUNOSORBENT ASSAY It was first described by Engvall and

Perlmann in 1971. It measures the amount of an Ab

present in a solution. Although it was first developed for the

measurement of Abs, it has since been modified for measurements of other molecules including hormones.

Both a competitive and a double Ab sandwich ELISA are available for performing Ag measurements, while Abs can be quantified by an indirect ELISA.

SANDWICH ELISA

The surface is first coated with a solid phase Ab.

The test sample, containing the Ag being measured, is then added and allowed to react with the bound Ab.

Any unbound Ag is washed away. A known amount of enzyme-

labelled Ab is then allowed to react with the bound Ag.

Any excess unbound enzyme-linked Ab is washed away after the reaction.

The substrate is then added and the reaction between the substrate and the enzyme produces a color change.

The amount of visual color change is a direct measurement of specific enzyme-conjugated bound Ab, and consequently Ag present in the specimen tested.

The "sandwich" technique is so called because the Ag being assayed is held between two different Abs.

COMPETITIVE ELISA FOR AG The test specimen containing the Ag to

be determined is mixed with a precise amount of enzyme-labelled Ag and both compete equally for a limited number of binding sites on an Ab found attached to the surface.

Excess free enzyme-labelled Ag is washed off before the substrate is added.

The key element in this competitive situation is the amount of color intensity resulting from substrate addition; the stronger the color intensity the less the Ag found in the sample.

COMPETITIVE ELISA FOR AG

ELISA FOR AB DETECTION (INDIRECT ELISA) The ELISA for the Ab detection is

very similar to the "sandwich Ag" technique, only instead of the Ab being adsorbed to the surface it is the Ag that is adsorbed.

The test sample containing the Ab is then added and allowed to bind to the attached Ag.

Unbound Ab is removed by several washings, after which enzyme-labelled Ag is added.

The enzyme-labelled Ag complex binds to a specific site on the bound Ab.

When a substrate is added, the enzyme-labelled Ag causes a color change by acting on the substrate.

The amount of color change is an indirect measure of specific Ab present in the test sample.

ELISA FOR AB DETECTION

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