inter-laboratory validation of a homologous elisa to quantify vitellogenin … · 2020. 3. 6. ·...

Biosense Laboratories ASInter-Laboratory validation FHM Vtg ELISA

Inter-Laboratory Validation of a homologousELISA to quantify Vitellogenin in the

fathead minnow (Pimephales promelas)

Janne K. Eidem, Hans Kleivdal and Anders Goksøyr

Biosense Laboratories ASThormøhlensgt 55N-5008 Bergenhttp://www.biosense.com

Contact:Janne K. Eidem+47 [email protected]


TABLE OF CONTENTS

SUMMARY 3INTRODUCTION 3

The fathead minnow Vitellogenin ELISA 5Brief summary of method 7

PURPOSE OF THE VALIDATION 7AIMS OF THE VALIDATION STUDY 8STUDY DESIGN 8

Participant Laboratories 8Sample preparation and spiking 9Sample and test kit shipments 9Data reporting and handling 9

VALIDATION RESULTS 10Applicability (Scope) 10Precision 10

Between-Day Precision RSDr 11Between-Laboratory Precision RSDR 12

Accuracy 12DISCUSSION 13CONCLUSION 15ACKNOWLEDGEMENTS 15REFERENCES AND LINKS 16APPENDIX 17

A. Single-Lab Validation ReportB. Letter to participantsC. Study protocol and report sheetD. Result sheetE. Individual raw data


SUMMARY

Vitellogenin (Vtg) is an established and sensitive endpoint for analysis of endocrinedisruption in fish. The widespread use of Vtg in this regard has led to the need forstandardised assays to quantify Vtg in fish samples. Fathead minnow (Pimephalespromelas) is an important fish test species used in ecotoxicology laboratories acrossthe world.

Based on monoclonal antibodies raised against fathead minnow (FHM) Vtg, wehave developed a quantitative sandwich enzyme-linked immunosorbent assay(ELISA) for FHM Vtg. A Single-Laboratory Validation has been performed earlier,according to international guidelines. Here, an Inter-Laboratory Validation study wascarried out in order to determine the performance of the FHM Vtg ELISA in fourdifferent laboratories. Study samples consisted of both plasma, whole bodyhomogenate (WBH) samples and ELISA kit Dilution buffer. The results aresummarised in Table 1, and show that the FHM Vtg ELISA is suitable forquantification of Vtg in both plasma and WBH samples from FHM in differentlaboratories.

Table 1: Summary of results from Inter-Laboratory Validation study of the FHM Vtg ELISA kit Performance characteristics Aim (%)1 Value (%)Between-Day Repeatability Precision, RSDr 16.4Between-Lab Reproducibility Precision, RSDR >50 18.6Recovery 50-200 69.4Bias -30.61Goksøyr et al 2003

***

INTRODUCTION

Vitellogenin (Vtg) is a large phospholipoglycoprotein, which functions as the egg yolkprecursor in oviparous vertebrates such as fish. The Vtg protein is produced in theliver and secreted from the liver cells through the secretory pathway, enters theblood circulation where it is transported to and taken up by growing oocytes.Endogenous oestrogen levels regulate Vtg production (Figure 1), and plasma Vtgconcentrations normally indicate the maturational status of the female fish (forreviews, see Mommsen & Walsh, 1988; Arukwe & Goksøyr, 2003). More than adecade ago, several studies demonstrated that also male fish, caught in rivers andstreams, had high levels of plasma Vtg (e.g. Purdom et al., 1994; Jobling et al.,1998), caused by chemicals present in the environment, acting like estrogens. Sincethen, numerous studies have shown the fish Vtg to be a highly responsivebiomarker for estrogenic compounds in both in vitro hepatocyte cell cultures, in vivoaquaria studies, and field studies (for reviews, see Kime, 1995; Sumpter & Jobling,1995; Arukwe & Goksøyr, 1998; 2003). Vtg induction in fish has now become anaccepted measure of xenoestrogenic potency of chemicals, effluents, anddischarges.


Vitellogenin Zona radiata P.

Estradiol

GonadsLiver

Figure 1: In response to oestradiol or xenoestrogens (x), Vitellogenin (egg yolk precursor) and zonaradiata proteins (egg shell precursors) are produced in the liver and transported via the blood to thegonads.

Within international bodies such as the Organization for Economic Cooperation andDevelopment (OECD), work is ongoing to develop screening and testingprogrammes for endocrine disrupting effects of new chemicals. In the focus of thisdevelopment are small fish test species including the fathead minnow (Pimephalespromelas), zebrafish (Danio rerio), and Japanese medaka (Oryzias latipes). Thesefish share several attributes that make them ideal test species for reproductivetoxicity testing, including small size at maturity, relatively short generation times,asynchronous spawning, and overall ease of culture.

Against this background, there is a need for specific, sensitive, and reliable methodsfor measuring Vtg levels in these fish species, assays that are readily available andgive reproducible results in different laboratories. The enzyme-linkedimmunosorbent assay (ELISA) is a sensitive laboratory technique widely used todetect and quantify antigens in a variety of biological samples. They can bequantitative (with a standard curve) or qualitative (semi-quantitative - without astandard curve). The two most widely used principles for quantitative detection ofproteins are the competitive ELISA and the sandwich ELISA techniques (Crowther,2001).***

X


THE FATHEAD MINNOW VITELLOGENIN ELISA

The Biosense fathead minnow (FHM) Vtg ELISA is based on a sandwich format,utilizing two different monoclonal mouse antibodies (Abs) raised against FHM Vtg.The antibodies were developed at the University of Florida, Gainesville, USA.

Figure 2: The principle of the fathead minnow Vtg ELISA.

The capture antibody is immobilised on ELISA microtiter plates, and binds to FHMVtg in the standard or sample added to the ELISA well (Figure 2). After unboundcomponents are washed away, a Detecting Ab, labelled with the enzymehorseradish peroxidase (HRP), is added. This Ab binds to a different part of the Vtgmolecule, creating a sandwich of antibodies and Vtg. Addition of the HRP substrateTetramethyl Benzidine (TMB) results in a colour reaction where the enzymecatalyses the conversion of this uncoloured substrate to a blue product. Afterdevelopment, the reaction is stopped by addition of a mild sulphuric acid, changingthe colour from blue to yellow. The colour intensity is measured using a microplatereader with a 450 nm filter, and is proportional to the concentration of Vtg in thestandard/sample.

All absorbance levels are corrected for non-specific background reading (NSB), anda calibration curve is created by plotting Vtg concentration on the x-axis and thecorresponding absorbance level on the y-axis (Figure 3). A 4 parameter or a log-logcurve fit can be used to describe the relationship between the concentration andthe signal. The equation for this calibration curve is then used to calculate the Vtgconcentration in plasma or WBH samples.***


FHM Vtg standard

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml

A4

50

-NS

B

0

10

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%C

V

Standard Within-Day Precision Between-Day Precision

Figure 3: Fathead minnow Vtg standard curve, compiled from 15 assays performed during theSingle-Laboratory Validation (using Microsoft Excel). The secondary y-axis shows the Within-Day andBetween-Day Repeatability Precisions (RSDr) for the standard curves.

A Single-Laboratory Validation of the FHM Vtg ELISA was performed at BiosenseLaboratories AS (Eidem et al 2005a). The validation demonstrated that the ELISA isboth sensitive, precise and reliable and thus fit for its intended purpose. Figure 3shows a representative standard curve derived from 15 assays performed duringthe Single-Lab Validation, including Between-Day and Within-Day RepeatibilityPrecision (RSDr) for the whole standard curve concentration range. Table 2summarises the results of the Single-Laboratory Validation. The complete resultsfrom this validation are presented in Appendix A.

Table 2: Summary of Single-Laboratory Validation results (Eidem et al 2005a) Performance characteristics Aim1 ValueSelectivity Matrix blank < LOD

(with the necessary dilutionfactor to avoid matrix effects)

No response at minimumdilution = 1:50 (plasma), 1:100(WBH)

Calibration Standard curve working range>10-fold, preferably 50-100fold to be practical with thedynamic range found in Vtglevels

Standard curve working range0.1-25 ng/ml (250-fold)

Accuracy (Recovery) Ideally 50-200% 75-106%Repeatability Precision RSDr <20% Within-Day RSDr: 4.5%

Between-Day RSDr: 9.9%Limit of Detection (LOD) <10 ng/ml 0.02 ng/ml (plasma)

0.04 ng/ml (WBH)Limit of Quantification (LOQ) <10 ng/ml 0.09 ng/ml (plasma)

0.11 ng/ml (WBH)Sample LOQ(=LOQ x necessary matrix dilution)

200 – 500 ng/ml 4.68 ng/ml (plasma, 1:50)11.35 ng/ml (WBH, 1:100)

Comparison with BiosenseCarp Vtg ELISA kit

R2>0.99

Comparison with competitorFHM Vtg ELISA kit

R2>0.99


BRIEF SUMMARY OF METHOD

Equipment and reagents required in addition to the FHM Vtg ELISA kit:• 0.3M H2SO4 (stop solution)• Microplate reader (wavelength 450 nm)• Pipettes with disposable tips (5-1000 µl)• Multi-channel pipette and reagent reservoir. Alternatively, a stepper pipette

with disposable tips (100 µl) can be used.• Test tubes (1-50 ml)• Microplate washing device (an automatic or manual plate washer is

recommended, but a squeeze bottle or a multichannel/stepper pipette canalso be used)

• Vortexer• Crushed ice

Summary of the ELISA method:1. Thaw samples on ice.2. Prepare dilutions of standard and samples on ice.3. To the pre-coated plates, add 100 µl Dilution buffer to the NSB wells.

Add 100 µl of diluted standards and samples to the remaining wells.Incubate at room temperature for 1.5 hour.

4. Wash the plates three times with 200 µl Washing buffer per well.Add 100 µl of diluted Detecting antibody to all wells.Incubate at room temperature for 0.5 hour.

5. Wash the plates five times with 200 µl Washing buffer per well.Add 100 µl Substrate solution to all wells.Incubate in the dark at room temperature for 20 minutes.

7. Add 100 µl of 0.3M H2SO4 to all wells to stop the reaction.8. Read the absorbance at 450 nm.9. Calculate the results.

Total sample capacity: 12 samples per plate (three dilutions of each sample,analysed in duplicates)Total assay time: 2 hours, 20 minutes.***

PURPOSE OF THE VALIDATION

In the process of evaluating Vtg as an endpoint for endocrine disruptor testing andscreening, various studies have been conducted over the recent years, involvingboth non-commercial laboratory methods and a few commercially available ELISAk i t s ( f o r z e b r a f i s h V t g i n t e r - c o m p a r i s o n s t u d y s e ehttp://abstracts.co.allenpress.com/pweb/setac2003/document/?ID=30012, for ano v e r v i e w o f v a l i d a t i o n s t a t u s i n t h e U S , s e ehttp://www.epa.gov/scipoly/oscpendo/assayvalidation/status.htm). These studies


have demonstrated the variance between different Vtg standards, between differentassays and between different laboratories using the same assay.

Clearly, there is a need for standardised, reproducible methods/kits that have beenthoroughly characterised, with validation data describing the assay’s performance,thus giving the end user reasonable expectancies. The aim of the following studywas to obtain inter-laboratory data on Precision and Accuracy, according tointernational guidelines (http://www.aoac.org).***

AIMS OF THE VALIDATION STUDY

In a 2003 document, Biosense took the initiative to stress the need for standardisedvalidations of Vtg standards and assays (Goksøyr et al, 2003). In this document,which was based on experience from participation in studies comparing Vtgmethods, a set of performance criteria was suggested, to be met by Vtgquantification methods (see under “Aims” in Table 1).

In this Inter-Laboratory Validation, a set of characteristics was analysed, chosen onthe basis of international guidelines from the AOAC (http://www.aoac.org). Theterminology involved in validation work is often confusing, and depends largely onwhich set of guidelines one looks at. Definitions used in this report is based onhttp://www.aoac.org/intaffairs/analytical_terminology.htm

This study aimed to obtain Inter-Laboratory Validaion data on the followingparameters:

1. Applicability (Scope)The analytes, matrices and concentrations for which a method of analysis may beused satisfactorily

2. Precision (Within-Laboratory, Between-Laboratory)Closeness of agreement between independent test results obtained understipulated conditions.

3. Accuracy (Recovery, Bias)Closeness of agreement between a test result and an accepted reference value.

***

STUDY DESIGN

PARTICIPANT LABORATORIES

In addition to Biosense Laboratories AS, three external laboratories were invited toparticipate in the validation. These laboratories have long, previous experience withthe ELISA technique, removing the lack of experience factor from the results.***


SAMPLE PREPARATION AND SPIKING

Sample blanks:

Plasma from male fathead minnows was purchased from Fish Soup, Newberry,Florida, USA. Plasma from 34 individual males was screened, and samples havingnon-detectable levels of Vtg at a 1:100 dilution were pooled.

WBH from male fathead minnows was a gift from Robert Bringolf, Iowa StateUniversity, USA. The WBH showed no detectable levels of Vtg at a 1:100 dilution.

Spiked samples:

For spiking/recovery studies, Sample blanks (plasma, WBH and kit Dilution buffer)were diluted 10-fold with kit Dilution Buffer containing different concentrations ofpurified, non-lyophilised Vtg. Samples were mixed thoroughly, aliquoted into suitablevolumes and frozen at –80oC. Concentrations in spiked samples were 5, 25 and125 ng/ml.

Naturally incurred samples:

Plasma with low and medium levels of Vtg was purchased from Fish Soup,Newberry, Florida, USA. Plasma from oestradiol (E2)-treated fish, as well as WBHwith low and medium levels of Vtg was a gift from Robert Bringolf, Iowa StateUniversity, USA. WBH from E2-treated FHM was prepared at BiosenseLaboratories. Samples were mixed thoroughly, aliquoted into suitable volumes andfrozen at –80oC.***

SAMPLE AND TEST KIT SHIPMENT

Aliquoted samples were shipped on dry ice using a courier company. Upon arrival,the participating laboratories verified that there was dry ice left in the container andthat the samples were in good (frozen) condition and that they were transferred to–80oC until analysis (see Report Sheet in Appendix C).

The ELISA kits were shipped on cool packs (not frozen) and were transferred to 4oCupon arrival.***

DATA REPORTING AND HANDLING

Each participating laboratory was given detailed instructions, plate layout and areport sheet into which the plate reader raw data was placed (see Result Sheet,Appendix C).

The data handling was checked with respect to the following (by study leader atBiosense Laboratories AS):

1. Standard curves were adjusted according to kit protocol.


2. Each standard and sample dilution had been performed in triplicates. If the CVbetween triplicate absorbance values was > 10%, the following was done:

• identify and remove a clear outlier to reduce CV to < 10%• if no clear outlier was identifiable, the whole data point was excluded

3. Sample dilutions with absorbance levels over or under the defined standardcurve working range were discarded.

4. If more than one sample dilution fell within the working range of the standardcurve, the average of these was calculated. If the results from these differentdilutions had a CV>50%, this data point was excluded.

***

VALIDATION RESULTS

APPLICABILITY (SCOPE)Applicability (Scope): The analytes, matrices and concentrations for which a methodmay be used satisfactorily.

This Inter-Laboratory Validation was performed in order to validate the performanceof the FHM Vtg ELISA, developed for use in endocrine disruptor screening tests.The same material was analysed by four different laboratories with previous ELISAskills. The ability of the ELISA to accurately analyse Vtg levels in spiked (fortified)plasma, WBH and the ELISA kit Dilution buffer was analysed. Four levels of Vtgwere used in this validation (0, 0.5, 2.5 and 12.5 ng/ml Vtg), corresponding todifferent levels within the standard curve working range (0.1-25 ng/ml). The followingvalidation data demonstrate that the FHM Vtg ELISA kit is suitable for Vtg analysis inthe matrices and in the concentrations ranges included in the validation.***

PRECISIONPrecision is the closeness of agreement between test results obtained under stipulatedconditions.• Repeatability Precision (same laboratory and operator, samples, equipment, short

time intervals), separated into Within-Day and Between-Day Repeatabilityprecision, usually expressed as relative standard deviation, RSDr.

• Reproducibility Precision (different laboratories, equipment and operators, samesamples). Usually expressed as relative standard deviation, RSDR.

Spiked and unspiked plasma and WBH sample blanks, as well as the ELISA kitDilution buffer, were analysed. On two different days, one aliquot was thawed,diluted 1:10 and analysed in triplicates. Unspiked plasma and WBH samples wereanalysed at three different dilutions according to kit protocol (plasma 1:50, 1:5000and 1:500000; WBH 1:100, 1:10000 and 1:1000000).


Table 3 shows the compiled data from all four laboratories and both days as well asBetween-Day Repeatability Precision RSDr.

***

Between-Day Repeatability Precision RSDr:

RSDr: Variation between results obtained for a given sample on the two different days.Average RSDr: Average RSDr for all samples analysed by one laboratoryOverall RSDr: Average RSDr for all samples and laboratories

Table 3: Between-Day Repeatability Precision RSDr Results from four laboratories, showingmeasured Vtg levels (ng/ml) and RSDr (%).

Lab1 Lab2 Lab3 Lab4code Day 1 Day 2 Average RSD

r

Day 1 Day 2 Average RSDr



PJ <w.r. <w.r. - - <w.r. <w.r. - - <w.r. <w.r. - - <w.r. <w.r. - -

PQ 0.4 0.4 0.4 2.1 0.3 0.3 0.3 1.8 0.4 0.4 0.4 6.4 0.5 nd 0.5 -

PB 2.0 2.0 2.0 1.9 1.4 1.6 1.5 10.0 2.1 2.1 2.1 0.8 nd 1.6 1.6 -

PS 10.0 10.5 10.3 3.1 8.5 10.4 9.4 14.5 13.0 12.5 12.7 2.8 8.1 10.1 9.1 15.7

WJ <w.r. <w.r. - - <w.r. <w.r. - - <w.r. <w.r. - - 1.0 <w.r. 1.0 -

WQ 0.3 0.3 0.3 13.3 0.2 0.3 0.2 17.7 <w.r. <w.r. - - <w.r. 0.2 0.2 -

WB 1.5 1.4 1.5 5.4 1.2 1.2 1.2 0.1 1.0 1.2 1.1 13.6 1.7 nd 1.7 -

WS 7.7 7.6 7.7 1.3 7.4 8.0 7.7 5.4 6.1 6.7 6.4 6.8 7.1 5.5 6.3 17.9

BJ <w.r. <w.r. - - <w.r. <w.r. - - <w.r. <w.r. - - <w.r. <w.r. - -

BQ 0.4 0.4 0.4 1.9 0.3 0.3 0.3 17.5 0.4 0.4 0.4 11.8 nd <w.r. - -

BB 2.0 1.9 2.0 5.2 1.8 1.8 1.8 1.7 2.2 1.9 2.1 8.2 <w.r. nd - -

BS 10.5 10.9 10.7 2.6 10.4 11.2 10.8 4.9 12.1 11.9 12.0 1.5 5.2 11.0 8.1 50.9

PM 952 1180 1066 15.2 1124 919 1021 14.2 1574 1192 1383 19.6 2289 849 1569 64.9

PA 38642 54454 46548 24.0 40192 37687 38939 4.6 57282 56422 56852 1.1 nd nd - -

PR 3940983 4883251 4412117 15.1 4084024 4374596 4229310 4.9 5273682 5116503 5195092 2.1 3935890 >w.r. 3935890 -

WM 7.92 <w.r. 7.92 - <w.r. <w.r. - - <w.r. <w.r. - - nd <w.r. - -

WA 27.0 22.4 24.7 13.3 26.3 13.1 19.7 47.1 <w.r. <w.r. - - nd <w.r. - -

WR 469214 516990 493102 6.9 477607 728601 603104 29.4 618288 478045 548166 18.1 nd 409530 409530 -

Average RSDr 7.9 12.4 7.7 37.4

Overall RSDr for all laboratories: 16.4

nd: not determined due to error or too high variance>w.r. and < w.r.: absorbance levels above or below working range of standard curve, respectively

Table 3 shows that the overall variation RSDr between results obtained on twodifferent days varied between 7.7 and 37.4% for the different laboratories. Theoverall RSDr for all four laboratories was 16.4%. Note that for Lab 4 RSDr was onlyobtainable for four samples.***


Between-Laboratory Reproducibility Precision RSDR:

RSDR: Variation between results obtained by different laboratories for one sample (averageof two days).Overall RSDR : Average RSDR for all laboratories and samples

Table 4: Between-Laboratory Reproducibility Precision RSDR. Results from four laboratories, showingmeasured Vtg levels (ng/ml, average of two days) and RSDR (%)code Average day1-2,

Lab1Average day1-2,Lab2

Average day1-2,Lab3

Average day1-2,Lab4

AverageLab1-4

RSDR

Lab 1-4PJ - - - - - -PQ 0.4 0.3 0.4 0.5 0.4 18.5PB 2.0 1.5 2.1 1.6 1.8 16.7PS 10.3 9.4 12.7 9.1 10.4 16.3WJ - - - - 1.0 -WQ 0.3 0.2 - - 0.3 15.0WB 1.5 1.2 1.1 1.7 1.3 17.7WS 7.7 7.7 6.4 6.3 7.0 12.3BJ - - - - - -BQ 0.4 0.3 0.4 - 0.4 15.6BB 2.0 1.8 2.1 - 1.9 7.4BS 10.7 10.8 12.0 8.1 10.4 21.0PM 1066 1021 1383 1569 1260 37.6PA 46548 38939 56852 - 47446 20.0PR 4412117 4229310 5195092 3935890 4515561 12.6WM 7.9 - - - 7.9 -WA 24.7 19.7 - - 22.2 28.7WR 493102 603104 548166 409530 528325 20.6

Overall Between-Laboratory RSDR 18.6

“-“ indicates that data was not obtainable for this sample (see Table 3)

Table 4 shows that the variation RSDR between results obtained in four differentlaboratories varied between 7.4 and 37.6% for the different samples. The overallRSDR for all samples was 18.6%.***

ACCURACYAccuracy (Trueness) is the closeness of agreement between a test result and anaccepted reference value. Recovery is the proportion of the amount of analyte, presentin or added to, the analytical portion, which is extracted and presented formeasurement. Bias is the difference between the test results and an accepted referencevalue.

Recovery and Bias in spiked samples were determined using the following formulas:

Recovery = (C1-C2)/C3 x 100Where C1= concentration measured in spiked sample, C2= concentration measured in unspikedsample, C3= theoretical concentration.


Bias = (C3-(C1-C2))/C3 x 100Where C1= concentration measured in spiked sample, C2= concentration measured in unspikedsample, C3= theoretical concentration

Table 5: Recovery and Bias. Results from analysis of spiked samples, showing measured Vtg levels(ng/ml, average of four laboratories), Recovery (%) and Bias (%)Sample type and code Theoretic conc.

(ng/ml)AverageLab1-4 (ng/ml)

RecoveryLab 1-4 (%)

Plasma PJ 0 - -PQ 0.5 0.4 74.8PB 2.5 1.8 73.3PS 12.5 10.4 83.0

WBH WJ 0 1.0 -

WQ 0.5 0.3 51.7WB 2.5 1.3 52.3WS 12.5 7.0 56.1

Buffer BJ 0 - -BQ 0.5 0.4 72.7BB 2.5 1.9 77.7

BS 12.5 10.4 83.2

Overall Recovery 69.4Bias -30.6

“-“ indicates that data was not obtainable for this sample (see Table 3)

The results show that the recovery and bias varied somewhat with both sample typeand spike concentration, with an overall Recovery of 69.4%. See Discussion formore on this. One lab reported a false positive on one day (sample WJ, Lab 4, seeTable 3).

***

DISCUSSION

An Inter-Laboratory Validation was performed to validate the performance of theBiosense FHM Vtg ELISA in four different laboratories. A set of 18 plasma, WBHand Buffer samples, comprising both spiked and unspiked samples, was analysedtwice, on two different days. Variation within laboratories (between days) andbetween laboratories was analysed, as well as the ability of the assay to measurecorrect values in spiked samples.

Kits and samples were successfully received and handled by participants, and datasets were obtained from all four laboratories.

Samples analysed on two different days determined the Between-Day RepeatabilityPrecision (RSDr). The RSDr varied from 7.7-37.4% for the different laboratories, withan overall RSDr of 16.4%. This value was elevated by a high variation and a low


number of RSDr values (only from four samples) from Lab 4. If the RSDr values ofthis laboratory are excluded, the overall RSDr is reduced to 9.4%.

Samples analysed by different laboratories allowed the determination of Between-Laboratory Reproducibility Precision (RSDR). The RSDR varied between 7.4 and37.6% for the different samples, and the overall RSDR for all samples was 18.6%.

Both the RSDr and the RSDR values are within the aims by Goksøyr et al (2003):RSDr < 20% and RSDR < 50%.

Analysis of spiked samples allowed the calculation of Recovery and Bias. plasma,WBH and ELISA kit Dilution buffer was spiked to three different concentrationscorresponding to low, medium and high concentration levels of the standard curve(working range = 0.1-25 ng/ml, see Single-Laboratory Validation, Appendix A/Eidemet al, 2005a). The Recovery varied somewhat with sample type and spike level: withDilution buffer giving the highest and WBH samples giving the lowest Recovery.Higher spike concentrations gave somewhat higher Recovery. An overall Recoveryof 69.4% was obtained, averaged over all sample concentrations and types. Thisvalue is within the suggested aim of 50-200% Recovery (Goksøyr et al. 2003).

Vtg is an unstable molecule, prone to degradation (Arukwe and Goksøyr 2003). Forthis reason, spiking of samples should ideally be done on the day of analysis toavoid freeze-thaw cycles and degradation. However, in order to obtain data fordetermination of Between-Day and Between-Laboratory Precision, samples had tobe diluted, aliquoted and frozen. This is likely to have affected the Recovery of Vtg inspiked samples. Spiking/recovery experiments performed in the Single-LaboratoryValidation, without freezing and thawing of the spiked samples, indicate recovery inboth plasma and WBH between 79-106% (see Table 2 and Single-LaboratoryValidation, Appendix A/Eidem et al, 2005a).

Some of the laboratories (especially Lab 4, but also Lab 2) reported high variancebetween replicate wells, caused by contamination of wells with enzyme-labelledDetecting Ab and a resulting unspecific colour development. Generally, one or moreof several factors can cause high variation between replicate wells:

• Imprecise pipettes and/or pipetting techniques• Imprecise washing techniques, or, in case of automatic plate washers,

uneven dispensing/aspiration• Careless handling of plates and plate sealers, which may result in splashing

and/or well-to-well contamination

Variation between replicate analyses of the same sample may often have the samereasons as variation between replicate wells. Variation is reduced by ensuringconsistent techniques when preparing standards, sample and antibody dilutions,when adding the solutions and when performing washing step. Also, keepingtemperatures and incubation times consistent is important. These factors should be


relatively easy to overcome with practise and proper maintenance of pipettes andequipment.

In addition to variation arising during performance of the assay, some variation isalso inherent in the assay itself. There will be some variation between differentplates, between different vials of Vtg standard RM and between different batches.However, these factors are low due to extensive quality controls during production.

Low absorbance was reported in obe case (Day 1, Lab 4), when a different washingbuffer than the one enclosed in the kit had been used – this wash buffer containedNa-azide as preservant, and which is known to inhibit HRP. On Day 2 the kit washbuffer was used, and higher absorbance was achieved.***

CONCLUSION

A successful Inter-Laboratory Validation was performed on the FHM Vtg ELISA kit.The results show that the kit performs with low variation and with good accuracyand that it is fit for its intended purpose, namely reliable quantification of Vtg inplasma or WBH samples from the fathead minnow (Pimephales promelas).***

ACKNOWLEDGEMENTS

Biosense Laboratories AS would like to thank the following participants for donatingtheir time and for their valuable input on the assay (in alphabetical order based oninstitution):

Dr. Nancy Denslow and Dr. Kevin J. Kroll University of Florida, Gainesville, USPhone: (352)-392-4700Fax: (352)-392-4707E-mail: [email protected]

Dr. Grace PanterAstraZeneca, Brixham Environmental LaboratoryBrixham, UKPhone: 01803 884261Fax: 01803882974E-mail: [email protected]

Dr. Charles Tyler and Dr. Ronny van AerleUniversity of ExeterExeter, UKPhone: 01392 264450Fax: 01392 263700E-mail: [email protected], [email protected] ***


REFERENCES AND LINKSAOAC “Harmonisation of analytical terminology in accordance with international standards”http://www.aoac.org/intaffairs/analytical_terminology.htm)

Arukwe A, Goksøyr A (1998). Xenobiotics, xenoestrogens and reproduction disturbances infish. Sarsia 83:225-241.

Arukwe A, Goksøyr A (2003). Eggshell and egg yolk proteins in fish: hepatic proteins for thenext generation. Oogenetic, population, and evolutionary implications of endocrinedisruption. Comp Hepatol 2: 4.

Brion F, Nilsen BM, Eidem JK, Goksøyr A, Porcher JM (2002). Development and validationof an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Daniorerio). Environ Toxicol Chem 28: 1699-1708.

Crowther JR (2001). The ELISA Guidebook. In: Methods Mol Biol 149. Humana Press,Totowa, NJ

Eidem JK, Kleivdal HK, Goksøyr A (2005a) Single-Laboratory Validation of a homologousELISA to quantify Vitellogenin in the fathead minnow (Pimephales promelas)(http://www.biosense.com/render.asp?ID=71&segment=3&session)

Goksøyr A. et al (2003) On the need for a standardized set-up for validation studies of fishvitellogenin assays as an endpoint in endocrine disruptor testing and screening – aproposal. http://www.biosense.com/Docs/GoksoyrEtal2003.pdf

Jobling S, Nolan M, Tyler CR, Brighty G, Sumpter JP (1998). Widespread sexual disruptionin wild fish. Environ Sci Technol 32: 2498–2506.

Kime DE (1995). The effects of pollution on reproduction in fish. Rev Fish Biol Fisheries5:52-96

Mommsen TP, Walsh PJ (1988). Vitellogenesis and oocyte assembly. In: Hoar WS, RandallVJ (eds) Fish Physiology XIA. Academic Press, New York, pp 347-406.

Nilsen B.M. et al 2004. Development of quantitative vitellogenin-ELISAs for fish test speciesused in endocrine disruptor screening. Anal Bioanal Chem 378:621http://www.springerlink.com, DOI 10.1007/s00216-003-2241-2

Norberg B, Haux C (1985). Induction, isolation and a characterization of the lipid content ofplasma vitellogenin from two Salmo species: rainbow trout (Salmo gairdneri) and sea trout(Salmo trutta). Comp Biochem Physiol 81B:869–876.

Porcher J-M. et al (2003). Intercomparison of zebrafish vitellogenin quantification methods.Society of Environmental Toxicology and Chemsitry, 24th annual meeting North America,http://abstracts.co.allenpress.com/pweb/setac2003/document/?ID=30012

Purdom CE, Hardiman PA, Bye VJ, Eno NC, Tyler CR, Sumpter JP (1994). Estrogeniceffects of effluents from sewage treatment works. Chem Ecol 8: 275-285.

Silversand C, Haux C (1995). Fatty acid composition of vitellogenin from four teleostspecies. J Comp Physiol 164B: 593-599.

Sumpter JP, Jobling S (1995). Vitellogenesis as a biomarker for estrogenic contamination ofthe aquatic environment. Environ Health Perspect 103 (Suppl 7): 173-178.

US EPA Endocrine Disruptor Screening Program, Assay status table.http://www.epa.gov/scipoly/oscpendo/assayvalidation/status.htm***


APPENDIX

A: SINGLE-LAB VALIDATION REPORT

Single-Laboratory Validation of a homologous ELISA to quantifyVitellogenin in the fathead minnow (Pimephales promelas)

Janne K. Eidem, Hans Kleivdal and Anders Goksøyr

Biosense Laboratories ASThormøhlensgt 55N-5008 Bergenhttp://www.biosense.com

Contact:Janne K. Eidem+47 [email protected]


TABLE OF CONTENTS

SUMMARY 3INTRODUCTION 4

The fathead minnow Vitellogenin ELISA 5Brief summary of method 6

PURPOSE OF THE VALIDATION 7AIMS OF THE VALIDATION STUDY 7

Definitions 8VITELLOGENIN STANDARD 9

Source for RM purification 10Purification of RM 10Quality assurance of RM 11Quantification of RM 12Stabilization of RM 13

SAMPLE PREPARATION AND SPIKING 14VALIDATION RESULTS 15

Applicability (Scope) 15Calibration 15LoD and LoQ (sensitivity) 16Selectivity (matrix effect) 18Repeatability Precision (intra- and inter-assay variation) 19Accuracy 22Ruggedness 23Comparison with existing methods 25

DISCUSSION 27CONCLUSION 28REFERENCES AND LINKS 29


SUMMARY

Vitellogenin (Vtg) is an established and sensitive endpoint for analysis of endocrinedisruption in fish. The widespread use of Vtg in this regard has led to the need forstandardised assays to quantify Vtg in fish samples. Fathead minnow (Pimephalespromelas) is an important fish test species used in ecotoxicology laboratories acrossthe world.Based on monoclonal antibodies raised against fathead minnow (FHM) Vtg, wehave developed a quantitative sandwich enzyme-linked immunosorbent assay(ELISA) for FHM Vtg. A Single-Laboratory Validation was performed according tointernational guidelines. Study samples consisted of both plasma, whole bodyhomogenate (WBH) and ELISA kit Dilution buffer. The results are summarised inTable 1, and show that the FHM Vtg ELISA is suitable for quantification of Vtg inboth plasma and WBH samples from FHM.

Table 1: Summary of results from Single-Laboratory Validation resultsPerformance characteristics Aim1 ValueSelectivity Matrix blank < LOD

(with the necessary dilutionfactor to avoid matrix effects)

No response at minimumdilution = 1:50 (plasma), 1:100(WBH)

Calibration Standard curve working range>10-fold, preferably 50-100 foldto be practical with the dynamicrange found in Vtg levels


Accuracy (Recovery) Ideally 50-200% 75-106%Repeatability Precision RSDr <20% Within-Day RSDr: 4.5%

Between-Day RSDr: 9.9%Limit of Detection (LOD) <10 ng/ml 0.02 ng/ml (plasma)

0.04 ng/ml (WBH)Limit of Quantification (LOQ) <10 ng/ml 0.09 ng/ml (plasma)

0.11 ng/ml (WBH)Sample LOQ(=LOQ x necessary matrix dilution)


Comparison with BiosenseCarp Vtg ELISA kit

R2>0.99

Comparison with competitorFHM Vtg ELISA kit

R2>0.99

1) Goksøyr et al 2003

***

INTRODUCTION

Vitellogenin (Vtg) is a large phospholipoglycoprotein, which functions as the egg yolkprecursor in oviparous vertebrates such as fish. The Vtg protein is produced in theliver and secreted from the liver cells through the secretory pathway, enters theblood circulation where it is transported to and taken up by growing oocytes.Endogenous oestrogen levels regulate Vtg production (Figure 1), and plasma Vtg


concentrations normally indicate the maturational status of the female fish (forreviews, see Mommsen & Walsh, 1988; Arukwe & Goksøyr, 2003). More than adecade ago, several studies demonstrated that also male fish, caught in rivers andstreams, had high levels of plasma Vtg (e.g. Purdom et al., 1994; Jobling et al.,1998), caused by chemicals present in the environment, acting like estrogens. Sincethen, numerous studies have shown the fish Vtg to be a highly responsivebiomarker for estrogenic compounds in both in vitro hepatocyte cell cultures, in vivoaquaria studies, and field studies (for reviews, see Kime, 1995; Sumpter & Jobling,1995; Arukwe & Goksøyr, 1998; 2003). Vtg induction in fish has now become anaccepted measure of xenoestrogenic potency of chemicals, effluents, anddischarges.

Vitellogenin Zona radiata P.

Estradiol

GonadsLiver

Figure 1: In response to oestradiol or xenoestrogens (x), Vitellogenin (egg yolk precursor) and zonaradiata proteins (egg shell precursors) are produced in the liver and transported via the blood to thegonads.

Within international bodies such as the Organization for Economic Cooperation andDevelopment (OECD), work is ongoing to develop screening and testingprogrammes for endocrine disrupting effects of new chemicals. In the focus of thisdevelopment are small fish test species including the fathead minnow (Pimephalespromelas), zebrafish (Danio rerio), and Japanese medaka (Oryzias latipes). Thesefish share several attributes that make them ideal test species for reproductivetoxicity testing, including small size at maturity, relatively short generation times,asynchronous spawning, and overall ease of culture.Against this background, there is a need for specific, sensitive, and reliable methodsfor measuring Vtg levels in these fish species, assays that are readily available andgive reproducible results in different laboratories. The enzyme-linkedimmunosorbent assay (ELISA) is a sensitive laboratory technique widely used todetect and quantify antigens in a variety of biological samples. They can bequantitative (with a standard curve) or qualitative (semi-quantitative - without astandard curve). The two most widely used principles for quantitative detection ofproteins are the competitive ELISA and the sandwich ELISA techniques (Crowther,2001).We have previously developed quantitative sandwich Vtg ELISAs for zebrafish andJapanese medaka, as well as a homologous carp Vtg ELISA, which also works well

X


for fathead minnow Vtg (Nilsen et al 2003). Here, we present Single-Laboratory (in-house) Validation data on a new, homologous quantitative sandwich ELISA formeasuring Vtg in plasma and whole body homogenate samples of the fatheadminnow. The validation has been carried out at Biosense, according to internationalguidelines for Single-Laboratory Validation (AOAC International Training Course,2003; Thompson et al, 2002; Eurachem, 1998).***

THE FATHEAD MINNOW VITELLOGENIN ELISA

The fathead minnow (FHM) Vtg ELISA is based on a sandwich format, utilizing twodifferent monoclonal mouse antibodies (Abs) raised against FHM Vtg. Theantibodies were developed at the University of Florida, Gainesville, USA.

Figure 2: The principle of the fathead minnow Vtg ELISA.

The capture antibody is immobilised on ELISA microtiter plates, and binds to FHMVtg in the standard or sample added to the ELISA well (Figure 2). After unboundcomponents are washed away, a Detecting Ab, labelled with the enzymehorseradish peroxidase (HRP), is added. This Ab binds to a different part of the Vtgmolecule, creating a sandwich of antibodies and Vtg. Addition of the HRP substrateTetramethyl Benzidine (TMB) results in a colour reaction where the enzymecatalyses the conversion of this uncoloured substrate to a blue product. Afterdevelopment, the reaction is stopped by addition of a mild sulphuric acid, changingthe colour from blue to yellow. The colour intensity is measured using a microplatereader with a 450 nm filter, and is proportional to the concentration of Vtg in thestandard/sample.

All absorbance levels are corrected for non-specific background reading (NSB), anda calibration curve is created by plotting Vtg concentration on the x-axis and thecorresponding absorbance level on the y-axis (Figure 3). A 4 parameter or a log-logcurve fit can be used to describe the relationship between the concentration andthe signal. The equation for this calibration curve is then used to calculate the Vtgconcentration in plasma or homogenate samples.


y = 0.1381x1.012

R2 = 0.9988

0.01

0.1

1

10

0.1 1 10 100

ng/ml Vtg

A450-N

SB

Figure 3: Fathead minnow Vtg standard curve***

BRIEF SUMMARY OF METHODEquipment and reagents required in addition to the FHM Vtg ELISA kit:

• 0.3M H2SO4 (stop solution)• Microplate reader (wavelength 450 nm)• Pipettes with disposable tips (5-1000 µl)• Multi-channel pipette and reagent reservoir. Alternatively, a stepper pipette

with disposable tips (100 µl) can be used.• Test tubes (1-50 ml)• Microplate washing device (an automatic or manual plate washer is

recommended, but a squeeze bottle or a multichannel/stepper pipette canalso be used)

• Vortexer• Crushed ice

Summary of the ELISA method:1. Thaw samples on ice.2. Prepare dilutions of standard and samples on ice.3. To the pre-coated plates, add 100 µl Dilution buffer to the NSB wells.

Add 100 µl of diluted standards and samples to the remaining wells.Incubate at room temperature for 1.5 hour.

4. Wash the plates three times with 300 µl Washing buffer per well.Add 100 µl of diluted Detecting antibody to all wells.Incubate at room temperature for 0.5 hour.

5. Wash the plates five times with 300 µl Washing buffer per well.Add 100 µl Substrate solution to all wells.Incubate in the dark at room temperature for 20 minutes.

7. Add 100 µl of 0.3M H2SO4 to all wells to stop the reaction.8. Read the absorbance at 450 nm.9. Calculate the results.


Total sample capacity: 12 samples per plate (three dilutions of each sample,analysed in duplicates)Total assay time: 2 hours, 20 minutes.***

PURPOSE OF THE VALIDATION

In the process of evaluating Vtg as an endpoint for endocrine disruptor testing andscreening, various studies have been conducted over the recent years, involvingboth non-commercial laboratory methods and a few commercially available ELISAk i t s ( f o r z e b r a f i s h V t g i n t e r - c o m p a r i s o n s t u d y s e ehttp://abstracts.co.allenpress.com/pweb/setac2003/document/?ID=30012, for ano v e r v i e w o f v a l i d a t i o n s t a t u s i n t h e U S , s e ehttp://www.epa.gov/scipoly/oscpendo/assayvalidation/status.htm). These studieshave demonstrated the variance between different Vtg standards, between differentassays and between different laboratories using the same assay.

Clearly, there is a need for standardised, reproducible methods/kits that have beenthoroughly characterised, with validation data describing the assay’s performance,thus giving the end user reasonable expectancies. The aim of the following studywas to obtain Single-Laboratory Validation data, according to internationalguidelines (AOAC, Eurachem, IUPAC).***

AIMS OF THE VALIDATION STUDY

In a 2003 document, Biosense took the initiative to stress the need for standardisedvalidations of Vtg standards and assays (Goksøyr et al, 2003). In this document,based on experience from participation in studies comparing Vtg methods, wesuggested a set of performance criteria to be met by Vtg quantification methods(Table 2).In this Single-Laboratory Validation (SLV), a set of characteristics were analysed,chosen on the basis of guidelines set up by international bodies like AOAC,Eurachem and IUPAC. It is recommended that “Single-Laboratory validationrequires the laboratory to select appropriate characteristics for evaluation from thefollowing: applicability, selectivity, calibration, accuracy, precision, range, limit ofquantification, limit of detection, sensitivity and ruggedness” (Thompson et al 2002).


Table 2 Pre-determined performance criteria (Goksøyr et al 2003)Performance characteristics AimSelectivity Matrix blank < LOD

(with the necessary dilution factor to avoid matrix effects)Calibration Standard curve working range >10-fold, preferably 50-100 fold

to be practical with the dynamic range found in Vtg levelsAccuracy1) Ideally 50-200%Repeatability 2) <20%Reproducibility3) <50%Limit of Detection (LOD) <10 ng/mlLimit of Quantification (LOQ) <10 ng/mlSample LOQ(=LOQ x necessary matrix dilution)

200 – 500 ng/ml

1) Referred to in this document as Recovery2) Referred to in this document Within-Day and Between-Day Repeatability Precision RSDr3) Referred to in this document Between-Laboratory Reproducibility Precision RSDR

This study aimed to obtain Single-Laboratory Validation data on the followingparameters:

4. Applicability5. Calibration6. Limit of detection (LOD), Limit of Quantification (LOQ)7. Selectivity8. Precision (Within-Day, Between-Day)9. Accuracy (spiking/recovery, bias)10. Ruggedness

In addition, comparisons with existing methods were also conducted.***

DEFINITIONSThe terminology involved in validation work is often confusing, and depends largelyon which set of guidelines one looks at. The following definitions have been used inthis report (see http://www.aoac.org/intaffairs/analytical_terminology.htm):Applicability (Scope): The analytes, matrices and concentrations for which a methodmay be used satisfactorily.Selectivity: The ability to measure accurately the analyte (Vtg) in the presence ofcomponents that may be expected to be present in the matrix (plasma and wholebody homogenate).Calibration, is the empirical determination of the relationship between the parametermeasured (e.g. ELISA absorbance) and the analyte (Vtg) concentration. The rangeof concentrations of analyte where such relationship is established is often referredto as "calibration range" or the "standard curve working range".Accuracy is the closeness of agreement between a test result and the acceptedreference value of the property being measured.


Recovery is the proportion of the amount of analyte, present in or added to, theanalytical portion, which is extracted and presented for measurementBias is the difference between the test results and an accepted reference valuePrecision is the closeness of agreement between test results obtained understipulated conditions.

• Repeatability Precision (same laboratory and operator, samples, equipment,short time intervals), separated into Within-Day and Between-DayRepeatability precision, usually expressed as relative standard deviation,RSDr.

• Reproducibility Precision (different laboratories, equipment and operators,same samples). Usually expressed as relative standard deviation, RSDR.

Limit of Detection is the smallest amount or concentration that can be reliablydistinguished from zero. Defined here as reagent blank + 3x standard deviation ofreagent blank. Indicates that the analyte is present, but not necessarily allowingexact quantification.Limit of Quantification A concentration above which the analytical method canoperate with an acceptable precision. Defined here as reagent blank + 10x standarddeviation of reagent blank.Sample LoQ. The LoQ corrected for minimum dilution factor necessary to avoidmatrix effect.Ruggedness. The ability of the measurement process to resist changes in resultswhen subjected to minor changes in environmental and procedural variables.***

VITELLOGENIN STANDARD

A validation of a quantitative Vtg ELISA should address not only the assay itself, butalso the Vtg standard used. Here we describe the purification, quality assuranceand quantification of the Vtg standard used in the FHM Vtg ELISA.

Important issues here are• the choice of source for purification• the purification method• the quality assurance• the quantification method• stabilization procedure

Ideally, a certified reference material (CRM) should be used as a standard inquantitative assays. In the case of a Vtg ELISA, this CRM should consist of intactVtg purified to apparent homogeneity from the test species in question, and shouldbe quantified according to accepted methods for protein quantification. In addition,the Vtg should be stabilized for shipping and storage.


Due to the lack of guidelines or criteria for these issues, we will not refer to the FHMVtg standard as CRM, but rather only as Reference Material (RM).***

SOURCE FOR RM PURIFICATION

Various sources of purified standards for Vtg assays have been used, includingplasma, WBH, liver homogenate, ascites fluid, and egg yolk. For each of thesesample types, the state of the Vtg will be different. For example, the liver cell willcontain immature (unprocessed) Vtg that has not undergone full post-translationalmodifications, as well as mature Vtg ready for secretion, whereas the egg yolk willcontain the lipovitellin form processed after uptake. WBH will contain a mixture ofthese (unless ovaries and /or liver have been removed), in addition to a high level ofproteolytic activity that may act to degrade the protein during preparation.

We chose to purify Vtg from plasma obtained from estrogenized FHM, becausesuch plasma contains intact, circulating Vtg at a high concentration (mg/ml levels).Plasma was kindly provided by Charles Tyler, University of Exeter, UK.

The matrix for RM production should be obtained from estrogenized fish exposed to17ß-oestradiol (or another given reference oestrogen) for a given period of time. Asuitable protease inhibitor (e.g. aprotinin or a protease inhibitor cocktail) should beadded to the matrix during sampling to avoid degradation of Vtg.***

PURIFICATION OF RM

Due to the instability of Vtg, the purification procedure should be as rapid aspossible while maintaining the integrity of the protein and yielding a pure product.Various methods include

• Ion exchange chromatography, with or without a following gel permeationclean-up (e.g. Brion et al., 2002).

• Selective precipitation of Vtg from plasma using MgCl2. This is a rapidmethod that has been successfully used with some species (e.g. trout andcarp: Norberg & Haux, 1985, and Nilsen et al., 2003), but appears to be lessuseful for other species.

• Immunoaffinity-based procedures (e.g. chromatography or magnetic beads).This strategy requires Vtg-specific Abs, and may give a bias in thecomposition of the purified product depending on the epitope specificity ofthe antibody used.

Different purification protocols may lead to different compositions of the Vtg holo-protein (i.e. different parts of the phospholipoglyco-modifications may be retained),which also may lead to different affinities of the antibodies in an ELISA. Using eitherof these methods, mg quantities of purified Vtg can be obtained within a shortperiod of time (10-30 min). To prevent degradation of the protein this process


should be performed in a cold environment using cold buffers containing proteaseinhibitors.We chose to use anion-exchange chromatography to purify the RM for the FHM VtgELISA. Using spin columns from Vivascience (Hannover, Germany), Vtg was rapidlypurified in sufficient quantities from small amounts of plasma from estrogenizedFHM. ***

QUALITY ASSURANCE OF RM

The purity and homogeneity of the RM needs to be established using gelelectrophoresis. Both SDS-PAGE and 2-dimensional electrophoresis (2-DE) werecarried out to demonstrate the purity of the RM (Figure 4). In addition, Matrixassisted laser desorption ionization mass spectrophotometry (MALDI-MS) wasperformed to obtain more information about the proteins observed on the 2-DE gel.

Both SDS-PAGE and 2-DE shows few impurities. In SDS-PAGE, three main bandsare clearly visible. All three bands are recognised by the FHM Vtg-specific mAb 1E9in western blot.Peptide sequencing of the spots “FHM1” and “FHM2”, obtained by MALDI-MS,were both recognised as FHM Vtg using the Mascot database (http://www.matrix-science.com). “FHM1” had hits in different parts of the protein, whereas “FHM2”only had hits in the N-terminal part, indicating that it is a breakdown product of themain 150 kDa Vtg holoprotein. “FHM3” was not recognised as Vtg, but must beseen as an unknown, low-molecular weight peptide contaminant.All in all, the results show that the purified protein is fathead minnow Vtg, and thatthe preparation is satisfactory pure.


a b

c

Figure 4 a: SDS-PAGE with different amounts of FHM Vtg. 7% separation gel. Lane 1: Broad rangestandard (Bio-Rad). Lane 2: Empty. Lane 3: 1 µg Vtg. Lane 4: 2 µg Vtg. Lane 5: 3 µg Vtg. Lane 6:Empty. Lane 7: 5 µg Vtg.Figure 4 b: Western blot. 1 µg Vtg was applied, and mAb 1E9 was used to detect the FHM VtgFigure 4 c: 2-DE of FHM Vtg. 50 µg Vtg was applied. pH 3-10, 9% separation gel, low rangestandard (Bio-Rad). The indicated proteins (FHM 1-3) were analysed with MALDI-MS.

***

QUANTIFICATION OF RM

Various methods are commonly used to quantify purified Vtg. All are dependent ona pure product.

• Staining methods such as Lowry or Bradford rely on the use of a proteinstandard, normally bovine serum albumin (BSA), and assume a similarstaining response of the protein in question to this standard protein. In manycases, this may not be true; especially for a protein like Vtg, which containsvarious non-peptide groups (sugars, phosphates, lipids).

25015010075

50

37

kDa 1 3 4 52 6 7

FHM3

FHM2

FHM1 97.4

66.2

kDa


• Measurement of absorbance at 280 nm is a simple and commonly usedquantification method. The method relies on the use of an extinctioncoefficient, which is different for each protein, due to the intramolecularenvironment affecting the exposure of UV absorbing aromatic amino acidresidues. A theoretical extinction coefficient can be calculated from the fullamino acid sequence of a protein, however posttranslational modificationswill affect this value.

• A more precise method of protein quantification is quantitative amino acidanalysis. This analysis can be performed by independent analyticallaboratories using standard instrumentation. The amino acid composition canbe compared to the theoretical composition if the sequence is known. Alimitation of this method is that only the protein portion of the Vtg molecule isquantified. The lipid and phosphate parts have been reported for somespecies to represent 15–20% and 0.6–0.8%, respectively (e.g. Silversand &Haux, 1995), whereas the carbohydrate portion is not well studied.

We feel that the reliability and independence of amino acid analysis outweighs thelimitations, and we chose this method for quantification of the FHM Vtg RM. Twoparallel analyses were performed at the Peptide Synthesis Lab at the BiotechnologyCentre of Oslo, Norway.***

STABILIZATION OF RM

Vtg is sensitive to freeze-thaw cycles (Figure 5), and needs to be stored at –80°Cand shipped on dry ice. In the Biosense laboratory, we have developed alyophilisation procedure that gives good stability to purified Vtg, with more than oneyear’s stability of Vtg at 4°C (these stability data are “real time” data, and are so farbased on other species than FHM). After lyophilisation, the RM Vtg was calibratedagainst non-lyophilised Vtg in the FHM ELISA.

Figure 5: Stability of lyophilised FHM Vtg during repeated freeze-thaw cycles. One vial ofreconstituted FHM Vtg was exposed to repeated freezing and thawing. The amount of Vtg in the vialwas analysed in the FHM Vtg ELISA.

FHM Vtg freeze/thaw cycles

0

20

40

60

80

100

0 1 2 3 4 5

Numer of freeze/thaw cycles

% o

f th

eo

reti

cal

am

ou

nt


The FHM Vtg RM was compared to FHM Vtg supplied as a standard for the USEPA method study (http://www.epa.gov/scipoly/oscpendo/assayvalidation/status.htm).This Vtg had been purified using anion-exchange chromatography and had alsobeen quantified using amino acid analyses, but the Vtg solution had been thawedonce before this comparison was made. Figure 6 shows that there are between 1.2and 1.6-fold difference between the absorbance values obtained with the twostandards. This small difference is most likely due to the state of degradation andpurity of the Vtg.

FHM standards

y = 0.2018x0.9864

R2 = 0.9992

y = 0.1514x0.9523

R2 = 0.9988

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml

A4

50

-NS

B

Biosense Standard

US EPA Standard

Power (Biosense Standard)

Power (US EPA Standard)

Figure 6: Comparison of the FHM Vtg RM with FHM Vtg supplied by the US EPA for Vtg assaycomparison study. The standards were analysed in the FHM Vtg ELISA.

***

SAMPLE PREPARATION AND SPIKING

Sample blanks:Plasma from male fathead minnows was purchased from Fish Soup, Newberry,Florida, USA. Plasma from 34 individual males was screened, and samples havingnon-detectable levels of Vtg at a 1:100 dilution were pooled.WBH from male fathead minnows was a gift from Robert Bringolf, Iowa StateUniversity, USA. The WBH showed no detectable levels of Vtg at a 1:100 dilution.Spiked samples:For spiking/recovery studies, Sample blanks (plasma, WBH and kit Dilution buffer)were diluted 10-fold with kit Dilution Buffer containing different concentrations ofpurified, non-lyophilised Vtg. Samples were mixed thoroughly, aliquoted into suitablevolumes and frozen at –80oC. Concentrations in spiked samples were 5, 25 and125 ng/ml.Naturally incurred samples:Plasma with low levels of Vtg was purchased from Fish Soup, Newberry, Florida,USA. WBH with low levels of Vtg was a gift from Robert Bringolf, Iowa StateUniversity, USA.***


VALIDATION RESULTS

APPLICABILITYApplicability (Scope): The analytes, matrices and concentrations for which amethod may be used satisfactorily.

This validation was performed in order to determine the characteristics of the FHMVtg ELISA, developed for analyses of Vtg in the fathead minnow (Pimephalespromelas), as an endpoint in endocrine disrupting chemicals (EDC) screening tests.The ability of the ELISA to accurately analyse Vtg levels in spiked (fortified) plasma,whole body homogenate (WBH) and the ELISA kit Dilution buffer was analysed.Four levels of Vtg were used in this validation (0, 0.5, 2.5 and 12.5 ng/ml Vtg),corresponding to different levels within the standard curve working range (0.1-25ng/ml).***

CALIBRATIONCalibration, is the empirical determination of the relationship between theparameter measured (e.g. ELISA absorbance) and the analyte (Vtg)concentration. The range of concentrations of analyte where such relationship isestablished is often referred to as "calibration range" or the "standard curveworking range".

Calibration of the assay was performed with purified, lyophilised FHM Vtg RM. Astandard serial dilution containing 11 concentration points was used, and a log-logcurve fit (using Microsoft Excel) was used to define the relationship betweenconcentration and response (absorbance).

Standard curves from 15 assays were compiled (Figure 6, Table 3 a-b). Threestandard curves were run each day for five days. The standard serial dilutions wereprepared fresh each day, and the same solution was used on three different ELISAplates. The Within-Day and Between-Day Precision was calculated (Figure 6).

FHM Vtg standard

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml

A4

50

-NS

B

0

10

20

30

40

50

60

70

80

90

100

%C

V

Standard Within-Day Precision Between-Day Precision

Figure 6: Combined FHM Vtg standard curve, average from 15 assays (using Microsoft Excel). Thesecondary y-axis shows the Within-Day and Between-Day Repeatability Precisions (RSDr) for thestandard curves.


Table 3a: Within-Day Repeatability Precision for FHM Vtg standard curvesWithin Day Precision

Concentration (ng/ml) NSB 0.05 0.10 0.20 0.39 0.78 1.56 3.13 6.25 12.50 25.00 50.00

Day 1 Average absorbance (450 nm),3 standard curves

0.061 0.069 0.072 0.084 0.108 0.167 0.276 0.507 0.918 1.822 3.338 5.307

%CV 8.2 11.1 8.7 5.3 4.2 4.4 4.0 4.5 4.4 2.2 2.8 16.1


0.060 0.065 0.072 0.083 0.110 0.166 0.278 0.506 0.940 1.811 3.194 5.793

%CV 9.0 5.9 5.3 6.2 8.7 10.2 10.1 11.5 11.0 10.0 8.7 7.3


0.057 0.062 0.069 0.079 0.102 0.153 0.255 0.460 0.849 1.657 3.027 5.263

%CV 5.3 3.3 5.7 3.6 3.2 4.6 6.5 6.6 5.7 5.8 5.2 13.9


0.055 0.060 0.066 0.074 0.094 0.140 0.215 0.416 0.735 1.444 2.657 4.358

%CV 5.6 5.0 5.9 2.3 3.7 5.8 5.1 6.3 5.2 5.1 4.2 12.4


0.056 0.063 0.069 0.082 0.112 0.170 0.271 0.498 0.945 1.661 3.009 5.017

%CV 4.1 2.1 1.6 4.8 4.4 6.4 8.4 9.4 7.6 11.5 3.8 17.4

Overall Within-Day Precision 6.4 5.5 5.4 4.4 4.8 6.3 6.8 7.7 6.8 6.9 4.9 13.4

Overall Within-Day Precision(working range, 0.1-25 ng/ml)

6.0

Table 3b: Between-Day Repeatability Precision for FHM Vtg standard curvesBetween-day Precision

Concentration (ng/ml) NSB 0.05 0.10 0.20 0.39 0.78 1.56 3.13 6.25 12.50 25.00 50.00

Average absorbance (450 nm), 5 days 0.058 0.064 0.070 0.080 0.105 0.159 0.259 0.477 0.877 1.679 3.045 5.148

%CV 4.2 5.1 3.8 5.1 6.8 7.8 10.0 8.3 10.1 9.1 8.4 10.2

Overall Between-Day Precision(working range, 0.1-25 ng/ml)

7.7

The results show that standard curves obtained on both the same day and ondifferent days show little variability, with an average Within-Day Precision of 6.0 %and a Between-Day Precision of 7.7 %.

The standard curve working range was between 0.1 and 25 ng/ml using a log-logtransformation of the data.***

LOD AND LOQ (SENSITIVITY)Limit of Detection is the smallest amount or concentration that can be reliablydistinguished from zero. Defined here as reagent blank + 3x standard deviation ofreagent blank. Indicates that the analyte is present, but not necessarily allowing exactquantification.

Limit of Quantification A concentration above which the analytical method canoperate with an acceptable precision. Defined here as reagent blank + 10xstandard deviation of reagent blank.


Sample LoQ. The LoQ corrected for minimum dilution factor necessary to avoid matrixeffect.

Limit of Detection (LoD) and Limit of Quantification (LoQ) were determined frommatrix blanks analysed during Precision studies. Since the samples analysed at a1:100 dilution did not have detectable levels of Vtg, LoD and LoQ were determinedfrom NSB-corrected absorbance levels and “translated” into concentration using therelevant standard curve equation (Table 4a-c).

Table 4a) LoD and LoQ for PlasmaReplicate 1A450nm

Replicate 2A450nm

Replicate 3A450nm

AverageA450nm

StdevA450nm

3x StdevA450nm

LoDng/ml

10x StdevA450nm

LoQng/ml

Run1 0.000 -0.003 -0.002 -0.001 0.002 0.003 0.02 0.014 0.11

Run2 0.001 -0.002 -0.003 -0.001 0.002 0.005 0.03 0.019 0.12

Run3 -0.001 -0.001 -0.001 -0.001 0.000 0.000 0.00 0.003 0.02

Run4 0.001 0.002 0.000 0.001 0.001 0.005 0.04 0.013 0.12

Run5 0.000 0.000 -0.003 -0.001 0.002 0.004 0.02 0.017 0.10

Average 0.003 0.02 0.013 0.09

Table 4b) LoD and LoQ for WBHReplicate 1A450nm

Replicate 2A450nm

Replicate 3A450nm

AverageA450nm

StdevA450nm

3x StdevA450nm

LoDng/ml

10x StdevA450nm

LoQng/ml

Run1 -0.004 0.000 -0.001 -0.002 0.002 0.005 0.04 0.020 0.15

Run2 -0.005 -0.006 -0.007 -0.006 0.001 -0.004 ND 0.002 0.01

Run3 -0.002 -0.004 -0.003 -0.003 0.001 -0.001 ND 0.005 0.04

Run4 0.002 -0.003 -0.005 -0.002 0.004 0.009 0.08 0.034 0.32

Run5 -0.002 -0.004 -0.003 -0.003 0.001 0.000 0.00 0.008 0.05

Average 0.002 0.04 0.014 0.11

Table 4c) LoD and LoQ for Dilution bufferReplicate 1A450nm

Replicate 2A450nm

Replicate 3A450nm

AverageA450nm

StdevA450nm

3x StdevA450nm

LoDng/ml

10x StdevA450nm

LoQng/ml

Run1 -0.002 0.001 -0.006 -0.002 0.004 0.009 0.06 0.033 0.24

Run2 -0.009 -0.008 -0.010 -0.009 0.001 -0.005 ND 0.003 0.02

Run3 0.000 0.001 -0.001 0.000 0.001 0.003 0.02 0.009 0.07

Run4 -0.001 -0.001 -0.001 -0.001 0.000 0.000 0.00 0.003 0.03

Run5 -0.002 -0.003 0.001 -0.001 0.002 0.005 0.03 0.019 0.11

Average 0.002 0.03 0.013 0.09

The LoD varied between 0.02 and 0.04 ng/ml for different sample types, and theLoQ varied between 0.09 and 0.11 ng/ml. Thus, the LoQ is in good agreement withthe lower limit of the standard curve working range (0.10 ng/ml).

***

SELECTIVITY (MATRIX EFFECT)

In order to determine the degree of interference from sample matrices onquantification of Vtg, plasma and WBH were investigated for adverse effects on thesignal response, i.e. plasma or matrix effect. Different dilutions of matrix blanks were


spiked with a range of Vtg concentrations, and recovery was measured andcompared.Figure 8 and 9 show that there is an inhibition of the signal at low dilutions of bothplasma and WBH, resulting in an underestimation of Vtg at these dilutions. Thiseffect varies somewhat between different samples, and with the spikeconcentration. Note that some haemolysis had happened during preparation of theplasma, which may have an effect on matrix effect.Based on these results showed in Figure 8 and 9, we recommend 1:50 for plasmaand 1:100 for WBH as the minimum dilution factors. Lower dilution factors might beused, but this should be tested in individual laboratories, and may depend on themethod of sample preparartion.

Parallelism between standard and unspiked samples

0.001

0.01

0.1

1

10

10 100 1000 10000 100000

(Relative) dilution factor (x-fold)

A4

50

-NS

B

Std

Plasma

Wbh

Wbh

Power(Plasma)Power (Std)

Power (Wbh)

Wbh

0.0

0.5

1.0

1.5

2.0

2.5

50 100 150 200 250 300 350 400 450 500 550

Dilution factor (x-fold)

A4

50

-NS

B

0 0.5 2.5 12.5

Plasma

0.0

0.5

1.0

1.5

2.0

50 100 150 200 250 300 350 400 450 500 550

Dilution factor (x-fold)

A4

50

-NS

B

0 0.5 2.5 12.5

Figure 8: Effect of plasma and WBH sample dilution on detection of Vtg. Experiments wereperformed with spiked samples (Figure 8a-b) or with naturally incurred samples (Figure 8c),containing Vtg.

a) b)

c)


Figure 9: Effect of plasma and WBH sample dilution on quantification of Vtg. Different amounts ofplasma or WBH were added to standard dilutions (Figure 9a-b). The average recovery for eachsample dilution factor was cacluated (Figure 9c). An additional experiment was conducted with evenlower dilution of plasma, tested with three different Vtg levels (Figure 9d).

***

REPEATABILITY PRECISION (INTRA- AND INTERASSAY VARIATION), RSDr

Precision is the closeness of agreement between test results obtained under stipulatedconditions.• Repeatability Precision (same laboratory and operator, samples, equipment, short

time intervals), separated into Within-Day and Between-Day Repeatabilityprecision, usually expressed as relative standard deviation, RSDr.

Matrix blanks from plasma and WBH, as well as ELISA kit Dilution buffer wereanalysed, spiked with three different concentrations of Vtg corresponding to thelow, medium and high parts of the standard curve working range. In addition,unspiked material was analysed. All samples had been aliquoted and stored at–80oC until analysis. On five successive days, three aliquots were thawed andanalysed in triplicates at a 1:100 final dilution.

Within-Day and Between-Day Repeatability Precision (RSDr), often referred to asintra- and interassay variation, were calculated (Table 5a-c and Table 6a-c).

Within-Day Repeatability Precision: %CV between the three aliquots analysed in one day.

Plasma

0.01

0.1

1

10

100

0.01 0.1 1 10 100

ng/ml (theoretical)

ng

/m

l (m

easu

red

STANDARD PLASMA 1:50 PLASMA 1:100 PLASMA 1:200

Wbh

0.01

0.1

1

10

100

0.01 0.1 1 10 100

ng/ml (theoretical)

ng

/m

l (m

easu

red

STANDARD + 5 µl WBH 1:50 WBH 1:100 WBH 1:200

Recovery vs dilution

0

20

40

60

80

100

120

50 100 200

Dilution factor

Avera

gw

e r

eco

very

(%

)

PlasmaWbh

Plasma

0

20

40

60

80

100

25 50 100

Dilution factor

Avera

ge r

eco

very

(%

)

Plasma

a) ba)

)

c) d)


Overall Within-Day Repeatability Precision: average of the individual Precision values for fivedays

Table 5a) Within-Day Precision for spiked Plasma samplesRun # Spike concentration

ng/mlReplicate 1ng/ml

Replicate 2ng/ml

Replicate 3ng/ml

Averageng/ml

RSDr%

1 0.5 0.3 0.3 0.3 0.3 0.7

2.5 1.9 1.8 1.9 1.9 3.0

12.5 10.8 10.5 10.8 10.7 1.5

2 0.5 0.3 0.3 0.3 0.3 2.6

2.5 1.8 1.7 1.7 1.7 3.4

12.5 9.9 9.4 9.2 9.5 3.7

3 0.5 0.4 0.3 0.3 0.3 8.8

2.5 2.0 1.9 1.9 1.9 3.7

12.5 10.9 10.3 10.7 10.6 2.8

4 0.5 0.4 0.4 0.3 0.4 5.8

2.5 2.1 1.9 1.9 2.0 4.0

12.5 12.6 11.3 11.8 11.9 5.3

5 0.5 0.3 0.3 0.3 0.3 7.8

2.5 1.6 1.7 1.6 1.6 3.1

12.5 9.9 10.0 9.8 9.9 0.6

Overall Within-Day Precision (%) 3.8

Table 5b) Within-Day Precision for spiked WBH samplesRun # Spike concentration

ng/mlReplicate 1ng/ml

Replicate 2ng/ml

Replicate 3ng/ml

Averageng/ml

RSDr%

1 0.5 0.3 0.3 0.3 0.3 4.6

2.5 1.6 1.5 1.5 1.6 4.1

12.5 8.0 7.2 7.4 7.5 5.5

2 0.5 0.3 0.3 0.3 0.3 4.7

2.5 1.5 1.5 1.5 1.5 3.3

12.5 7.9 7.4 7.5 7.6 3.4

3 0.5 0.3 0.3 0.3 0.3 4.4

2.5 1.8 1.9 1.8 1.8 2.4

12.5 9.1 9.5 9.5 9.4 2.6

4 0.5 0.4 0.3 0.3 0.4 7.0

2.5 2.0 1.7 1.8 1.8 7.7

12.5 10.3 9.0 9.6 9.7 6.5

5 0.5 0.3 0.3 0.3 0.3 10.1

2.5 1.9 1.7 1.7 1.8 7.3

12.5 9.6 9.1 8.8 9.2 4.3


Table 5c) Within-Day Precision for spiked Dilution buffer samples


Run # Spike concentrationng/ml

Replicate 1ng/ml

Replicate 2ng/ml

Replicate 3ng/ml

Averageng/ml

RSDr%

1 0.5 0.5 0.4 0.4 0.4 10.3

2.5 2.1 2.2 2.1 2.1 2.3

12.5 11.1 11.1 11.3 11.1 0.9

2 0.5 0.3 0.3 0.3 0.3 3.7

2.5 1.8 1.8 1.9 1.9 2.5

12.5 11.2 10.6 10.3 10.7 4.2

3 0.5 0.4 0.4 0.4 0.4 3.1

2.5 2.2 2.2 2.2 2.2 1.2

12.5 11.4 11.1 11.4 11.3 1.9

4 0.5 0.4 0.4 0.4 0.4 2.4

2.5 2.3 2.3 2.4 2.3 2.3

12.5 11.6 11.7 12.2 11.8 2.6

5 0.5 0.4 0.4 0.3 0.4 4.2

2.5 2.2 2.1 1.5 2.0 20.8

12.5 12.3 11.4 11.0 11.6 5.9


Overall Within-Day Precision for all sample types and concentrations 4.5

Between-Day Repeatability Precision: % CV between the average measured concentrationsfrom each day.Overall Between-Day Repeatability Precision: Average Precision for all concentrations

Table 6a) Between-Day Precision for Plasma samplesSpike concentrationng/ml)

Average(ng/ml)

RSDr

%0.5 0.3 11.5

2.5 1.8 8.2

12.5 10.3 8.8

Overall Between-Day Precision 9.5

Table 6b) Between-Day Precision for WBH samplesSpike concentrationng/ml)

Average(ng/ml)

RSDr

%0.5 1.8 1.9

2.5 1.6 10.3

12.5 5.8 17.4

Overall Between-Day Precision 9.9


Table 6c) Between-Day Precision for Dilution buffer samplesSpike concentrationng/ml)

Average(ng/ml)

RSDr

%0.5 0.4 14.7

2.5 2.1 11.4

12.5 11.1 4.7

Oerall Between-Day Precision 10.3

Overall Between-Day Precision for all sample types and concentrations 9.9

The results show low variation in quantification, both within the same day (OverallWithin-Day Precision = 4.5 %) and between successive days (Overall Between-DayPrecision = 9.9 %).***

ACCURACYAccuracy is the closeness of agreement between a test result and the acceptedreference value of the property being measured.

Recovery is the proportion of the amount of analyte, present in or added to, theanalytical portion, which is extracted and presented for measurement

Bias is the difference between the test results and an accepted reference valueRecovery and bias

The concentrations measured in the spiked samples during Precision studies werecompared to the theoretical values and Recovery and Bias were determined usingthe following formulas (Table 7-8):

Recovery = (C1-C2)/C3 x 100Where C1= concentration measured in spiked sample, C2= concentration measured in unspikedsample, C3= theoretical concentration.

Bias = (C3-(C1-C2))/C3 x 100

Where C1= concentration measured in spiked sample, C2= concentration measured in unspikedsample, C3= theoretical concentration

Table 7: Recovery (expressed as % of theoretical concentration)Spike concentration (ng/ml) Plasma WBH Spiked buffer

0.5 64.2 63.3 75.9

2.5 72.8 68.0 83.9

12.5 84.3 69.4 90.5

Overall Recovery (%) 73.8 66.9 83.4

Overall Recovery for all sample types and concentrations (%) 74.7


Table 8: Bias (expressed as % difference from theoretical value)Spike concentration (ng/ml) Plasma WBH Spiked buffer

0.5 -35.8 -36.7 -24.1

2.5 -27.2 -32.0 -16.1

12.5 -15.7 -30.6 -9.5

Overall Bias (%) -26.3 -33.1 -16.6

Overall Bias for all sample types and concentrations (%) -25.3

The results show that the recovery and bias depend on both sample type and spikeconcentration, with an overall 25% underestimation of spike concentrations in thesample.Using freshly spiked samples, without previous freezing/thawing of the samples,Recovery was measured during the Selectivity study (see Figure 9). Results fromthese experiments, using several different concentrations of Vtg (standard curvedilution series), and different dilutions of plasma and WBH, gave the Recoveryresults much closer to 100% (Table 9a-c).

Table 9a: Recovery in WBH and plasma: Average Recovery for 11 different spike concentrations(expressed as % of theoretical value)Dilution factor Plasma WBH

1:50 81.4 102.0

1:100 106.7 102.0

1:200 97.6 105.9

Table 9b: Recovery in WBH and plasma: Average Recovery for three different spike concentrations(expressed as % of theoretical value)Dilution factor Plasma WBH

1:25 65.6 nd

1:50 75.9 84.8

1:100 95.2 102.5

Table 9c: Recovery in WBH and plasma, average of two experiments (see Table 9a and b)Dilution factor Plasma WBH

1:25 65.6 nd

1:50 78.7 93.4

1:100 101.0 102.3

1:200 97.6 105.9

***

RUGGEDNESSRuggedness. The ability of the measurement process to resist changes in resultswhen subjected to minor changes in environmental and procedural variables.

In order to investigate the stability of the assay when exposed to variations in theenvironment and assay procedure, seven parameters were combined in eightassays to determine their effect on quantification in the FHM Vtg ELISA (accordingto Youden & Steiner, 1975).The effect on quantification of the three matrix blanks, spiked with three differentconcentrations, was analysed. Table 10 shows the compiled results.


Table 10: Ruggedness test. Standard conditions are shown in bold. Alterations higher than 10% arehighlighted.

Sample concentration levelCondition altered Value ofcondition

Sample type

low med high

Difference (%) 1 Average (%) 1

Buffer temperature Cold

Buffer 7 6 4 6

Plasma -11 -10 -13 -11

RT

WBH 4 -9 -12 -5

Incubation temperature RT2

30oC Buffer 12 11 4 9

Plasma -18 -18 -21 -19

WBH -7 -5 -8 -7

Standard/sample incubation time 1.5 h

1 h Buffer -2 1 7 2

Plasma 6 7 11 8

WBH 1 4 8 4

Detection Ab incubation time 0.5 h

1 h Buffer 5 4 -3 2

Plasma -1 -10 -11 -7

WBH 3 1 -5 0

Number of washes beforedevelopment

5

3 Buffer 8 3 -6 2

Plasma 16 11 -1 9

WBH 11 7 -5 4

TMB solution temperature RT

4oC Buffer 7 0 1 3

Plasma 9 8 4 7

WBH 4 3 1 3

Development time 20 min

30 min Buffer -8 -5 1 -4

Plasma -2 1 6 2

WBH -7 -4 0 -3

1 Minus sign denotes that the value for the unaltered condition was lower than for the alteredcondition2 RT in this assay was 23oC

The results show that buffer and incubation temperature have the highest influenceon Vtg quantification in the FHM Vtg ELISA. The effect of buffer temperature wasstudied in separate assays, and showed that room-tempered buffer had a strongereffect on the samples than on the standard, increasing absorbance and themeasured Vtg concentration in the samples (data not shown). These resultsdemonstrate the importance of keeping the Dilution buffer cold and to keep theroom temperature relatively low (20-25oC). On the other hand, deviations to theincubation times have less influence on the quantification of samples.

***


COMPARISON WITH EXISTING METHODS

No established reference method for measuring FHM Vtg exists today, so the newFHM Vtg ELISA was therefore compared with two other commercially availableELISA kits, a Carp Vtg ELISA (Biosense Laboratories) and a FHM Vtg ELISA(competitor).***Carp Vtg ELISA (Biosense Laboratories AS)Plasma and WBH samples from the US EPA FHM Vtg assay comparison study(2003) were tested in the FHM Vtg ELISA. These samples had previously beentested in the Biosense Carp Vtg ELISA, using both the kit (carp) Vtg standard andthe FHM Vtg standard supplied with the study samples. Figure 10a and 10d showthe results compared. The Carp Vtg ELISA utilises one monoclonal and onepolyclonal carp Vtg-specific antibodies, as well as carp Vtg standard, and the assayis therefore heterologous for the FHM. However, the antibodies show excellentcross-reactivity with FHM Vtg (Nilsen et al 2004), reflected in values varying lessthan 2-fold between the two assays and a good correlation between Vtg levelsmeasured in the samples (R2 > 0.99).***Competitor FHM ELISA kitThe Biosense and a competitor FHM Vtg ELISA kit were compared using spikedand unspiked samples with different Vtg levels. Figure 10b, c and e show the twostandard curves and the results from the analysed samples. Although the absolutevalues varies on average less than 2-fold, due to differences in standards andantibodies, the results show very good correlation (R2 > 0.99). The largest differenceis the sensitivity of the assays, with a 78 times more sensitive standard curve in theBiosense FHM ELISA, and a corresponding lack of detection of Vtg in samplesspiked with low Vtg concentrations in the competitor ELISA kit.***


Figure 9: Comparison of the Biosense FHM Vtg kit with two methods. Figure 9a: Samples from a USEPA method comparison study were compared in the Biosense Carp (C) and FHM Vtg ELISA kits,using both C and FHM Vtg standard in the C ELISA. Figure 9b: FHM Vtg kit standard curves in theBiosense and competitor FHM Vtg ELISA kit. Figure 9c: Identical samples analysed in the Biosenseand competitor FHM Vtg ELISA kit. 9d: Correlation between results obtained with Biosense Carp andFHM Vtg ELISA kits. 9e: Correlation between Biosense and competitor FHM Vtg ELISA kits.

***

Standard curves

y = 0.1443x1.0271

R2 = 0.9986

y = 0.0039x1.0359

R2 = 0.99860.001

0.01

0.1

1

10

0.01 0.1 1 10 100 1000

ng/ml Vtg

A4

50

-NS

B Biosense

Competitor

Power (Biosense)

Power (Competitor)

b)

Plasma samples

0.1

1

10

100

1000

10000

100000

1000000

Sample ID

µg

/m

l in

pla

sma

FHM ELISA 4488 463.9 303.3 6943 0.427 0.272 0.222 0.399 0.255 4418 49773 48807 52006 507.4

C ELISA, C standard 6719 169.0 0.326 522.5 6388 25156 0.252 0.209 0.253 0.310 20073 30526 0.259 6727 67459 85811 58270 318.8

C ELISA, FHM standard 10893 344.8 0.552 740.7 10385 34537 0.434 0.371 0.443 0.525 27518 41963 0.450 10904 95532 12148 82559 631.6

A B D E G H I K L M N P R S T U W Y

a)

Biosense FHM Vtg ELISA vs competitor FHM ELISA

0.1

1

10

100

1000

10000

100000

1000000

10000000

ng

/m

l

Biosense

Competitor

Biosense 0.36 2.12 11.4 0.30 1.54 8.66 0.35 2.16 11.4 6.89 25.2 45011 996 38334 46661

Competitor 21.5 14.63 36.9 84219 1956 84441 74427

PJ PQ PB PS WJ WQ WB WS BJ BQ BB BS WM WA WR PM PA PR

c)

R2 = 0.9911

R2 = 0.9937

0.1

1

10

100

1000

10000

100000

0.1 1 10 100 1000 10000 100000

FHM kit

Car

p k

it Wbh samples

Plasma samples

Power (Wbh samples)

Power (Plasma samples)

R2 = 0.9988

0.1

1

10

100

1000

10000

100000

1000000

10000000

0.1 1 10 100 1000 10000 100000 1E+06 1E+07

Biosense kit

Com

pet

itor

kit

d)e)


DISCUSSION

A new sandwich ELISA, based on monoclonal antibodies raised against FHM Vtg,has been developed to quantify Vtg in samples from FHM. Vtg was purified fromFHM plasma to be used as standard in the ELISA,. The Vtg was furthercharacterised and quantified, and was found to be of sufficient purity to be usedreliably as Reference Material (RM) in the FHM ELISA.

A Single-Laboratory Validation was performed to validate the performance of thenew Biosense FHM Vtg ELISA. A set of plasma, WBH and ELISA kit Dilution buffersamples, both spiked, unspiked and naturally incurred (containing Vtg), wereanalysed on different days. Variation within days and between days wasdetermined, as well as the ability of the assay to measure correct values in spikedsamples.

A calibration curve was run, prepared from a serial dilution of the FHM Vtg RM, ineach assay. Using log-log transformation of the standard curves, the working rangewas from 0.1-25 ng/ml (250-fold). For 15 standard curves (three separate standardcurves per day for five days), the Within-Run and Between-Run RepeatabilityPrecision (RSDr) within the working range was 6.0 and 7.7%, respectively. Thisshows that the variability between different vials of Vtg RM is low, as is the variabilitybetween standard curves prepared on different days. Also, the 250-fold workingrange is well within our aim (100-fold, Goksøyr et al, 2003) Vtg concentrations insamples can vary over several orders of magnitude, and a broad working range isimportant because fewer dilutions from each sample of unknown Vtg concentrationare necessary in order to “hit” the standard curve working range.

Important parameteres for a quantitative assay is the Limit of Detection (LoD) andLimit of Quantification (LoQ). The FHM assay is highly sensitive, with an average LoDof 0.03 ng/ml and an LoQ equal to the lower limit of the standard curve workingrange, 0.10 ng/ml. With minimum recommended sample dilutions of 1:50 forplasma and 1:100 for WBH, the sample LOQs were 4.7 and 11.4 ng/ml,respectively. Both plasma and WBH from unexposed male fish (from the US EPAStudy) were easily quantified by the FHM ELISA.

In order to determine the Within-Day and Between-Day Repeatability Precision(RSDr) of the ELISA, three replicates of each sample were analysed on five differentdays. Within-Day RSDr values were between 3.8 and 4.6 % for the different sampletypes, with an overall RSDr of 4.5%. The Between-Day RSDr was between 9.5 and10.3%, with an overall RSDr of 9.9%. These values show that the assay shows littlevariability between runs, and are well within the aims defined by Goksøyr et al(2003).

The assay’s ability to accurately quantify Vtg in spiked samples was analysed bycomparing concentrations measured in the ELISA with the theoreticalconcentrations. Samples with three different concentrations of Vtg were analysed,


and the Recovery varied between 63 and 91%, depending on concentration andsample type, with an overall Recovery of 75% (Bias = -25%).Vtg is an unstable molecule, prone to degradation (Arukwe and Goksøyr 2003). Forthis reason, spiking of samples should ideally be done on the day of analysis toavoid freeze-thaw cycles and degradation. However, in order to obtain data fordetermination of Between-Day Repeatability Precision, samples had to be diluted,aliquoted and frozen. This is likely to have affected the Recovery of Vtg in spikedsamples. Spiking/recovery experiments performed without freezing and thawing ofthe spiked samples, gave Recovery in both plasma and WBH between 79-106%,supporting this theory.The Ruggedness test revealed that small changes to factors such as incubationtime and temperatures affected the FHM ELISA’s ability to quantify Vtg to differentdegrees. Seven factors were modified and tested together in different combinationsaccording to Youden & Steiner (1975), and the factors having highest impact on Vtgquantification were elevated buffer and room temperatures.

Since no official Reference Method for FHM Vtg quantification is established, thenew Biosense FHM ELISA was compared to two exisiting Vtg ELISA kits. TheBiosense Carp Vtg ELISA, although heterologous to the FHM, has successfullybeen applied on samples from FHM (Nilsen et al, 2004). A set of FHM samples fromthe US EPA method comparison study (2003) was re-analysed in the FHM ELISA.The absolute values obtained with the two kits differed less than 2-fold, and thecorrelation (R2) between the results were >0.99, showing that the two kits givecomparable results. An alternative FHM ELISA kit was also tested, using a set ofspiked and unspiked samples. The Biosense FHM kit standard curve was 78-foldmore sensitive than the competitor ELISA Vtg standard, but the absolute valuesvaried less than 2-fold and the correlation between the results from the two kits wasequally good, with R2>0.99.

The results from these two comparisons show that although absolute values maydiffer, due to factors such as differences in antibodies, Vtg standard quantification,purity, the assays still yield similar correlating results and suit their purpose ofdifferentiating between control and exposed groups of fish in studies of endocrinedisruption.

***

CONCLUSION

A successful Single-Laboratory Validation was performed on the Biosense FHM VtgELISA. The results, summarised in Table 11, show that our pre-defined aims(Goksøyr et al 2003) were met with good margin. The kit is sensitive and reliable,and is therefore a good tool which fits its purpose for quantitative analysis of Vtg inthe fathead minnow.


Table 11: Summary of Single-Laboratory Validation results for the Biosense FHM Vtg ELISA kitPerformance characteristics Aim1 ValueSelectivity Matrix blank < LOD

(with the necessary dilution factorto avoid matrix effects)

No response at minimum dilutionMinimum dilution =1:50 (plasma), 1:100 (WBH)

Calibration Standard curve working range>10-fold, preferably 50-100 foldto be practical with the dynamicrange found in Vtg levels


Accuracy (Recovery) Ideally 50-200% 75%2)

79-106%3)

Repeatability4) <20% Within-Day RSDr: 4.5%Between-Day RSDr: 9.9%

Limit of Detection (LOD) <10 ng/ml 0.02 ng/ml (plasma)0.04 ng/ml (WBH)0.03 ng/ml (buffer)

Limit of Quantification (LOQ) <10 ng/ml 0.09 ng/ml (plasma)0.11 ng/ml (WBH)0.09 ng/ml (buffer)

Sample LOQ(=LOQ x necessary matrix dilution)


1) Goksøyr et al 20032) From Precision studies, samples frozen and thawed once, dilution 1:1003) From Selectivity studies, samples freshly spiked and not frozen, dilutions 1:50-1:1004) Referred to in this document Within-Day and Between-Day Repeatability Precision, RSDr

***

REFERENCES AND LINKSAOAC “Harmonisation of analytical terminology in accordance with international standards”http://www.aoac.org/intaffairs/analytical_terminology.htm)

Arukwe A, Goksøyr A (1998). Xenobiotics, xenoestrogens and reproduction disturbances infish. Sarsia 83:225-241.

Arukwe A, Goksøyr A (2003). Eggshell and egg yolk proteins in fish: hepatic proteins for thenext generation. Oogenetic, population, and evolutionary implications of endocrinedisruption. Comp Hepatol 2:4.

Brion F, Nilsen BM, Eidem JK, Goksøyr A, Porcher JM (2002). Development and validationof an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Daniorerio). Environ Toxicol Chem 28: 1699-1708.

Crowther JR (2001). The ELISA Guidebook. In: Methods Mol Biol 149. Humana Press,Totowa, NJ

Goksøyr A. et al (2003) On the need for a standardized set-up for validation studies of fishvitellogenin assays as an endpoint in endocrine disruptor testing and screening – aproposal. http://www.biosense.com/Docs/GoksoyrEtal2003.pdf

Jobling S, Nolan M, Tyler CR, Brighty G, Sumpter JP (1998). Widespread sexual disruptionin wild fish. Environ Sci Technol 32: 2498–2506.


Kime DE (1995). The effects of pollution on reproduction in fish. Rev Fish Biol Fisheries5:52-96

Mommsen TP, Walsh PJ (1988). Vitellogenesis and oocyte assembly. In: Hoar WS, RandallVJ (eds) Fish Physiology XIA. Academic Press, New York, pp 347-406.

Nilsen B.M. et al 2004. Development of quantitative vitellogenin-ELISAs for fish test speciesused in endocrine disruptor screening. Anal Bioanal Chem 378:621http://www.springerlink.com, DOI 10.1007/s00216-003-2241-2

Norberg B, Haux C (1985). Induction, isolation and a characterization of the lipid content ofplasma vitellogenin from two Salmo species: rainbow trout (Salmo gairdneri) and sea trout(Salmo trutta). Comp Biochem Physiol 81B:869–876.

Porcher J-M. et al (2003). Intercomparison of zebrafish vitellogenin quantification methods.Society of Environmental Toxicology and Chemsitry, 24th annual meeting North America,http://abstracts.co.allenpress.com/pweb/setac2003/document/?ID=30012

Purdom CE, Hardiman PA, Bye VJ, Eno NC, Tyler CR, Sumpter JP (1994). Estrogeniceffects of effluents from sewage treatment works. Chem Ecol 8: 275-285.

Silversand C, Haux C (1995). Fatty acid composition of vitellogenin from four teleostspecies. J Comp Physiol 164B: 593-599.

Single Laboratory Validation of Analytical Methods for Dietary Supplements (2003). AOACInternational Training Course in Atlanta, Georgia, USA

Sumpter JP, Jobling S (1995). Vitellogenesis as a biomarker for estrogenic contamination ofthe aquatic environment. Environ Health Perspect 103 (Suppl 7): 173-178.

The Fitness for Purpose of Analytical Methods (1998). Eurachem,http://www.eurachem.ul.pt/guides/valid.pdf

Thompson et al, 2002. Harmonized guidelines for Single-Laboratory validation of methodsof analysis. IUPAC Technical Report, Pure Appl Chem 74 (5): 835-855

US EPA Endocrine Disruptor Screening Program, Assay status table.http://www.epa.gov/scipoly/oscpendo/assayvalidation/status.htm

Youden & Steiner (1985). In Use of statistics to develop and evaluate analytical methods.AOAC International, Gaithersburg, MD.

***


B: LETTER TO PARTICIPANTS

Dear Colleague,

Bergen, December 6th, 2004

Validation material – Fathead minnow Vtg ELISA kit and samples

We hope that the material arrived safely and in time. I would like you to do the followingimmediately upon receipt - please note:

1. The state of the samples – samples were shipped on dry ice. There should be dry ice leftin the box, and the samples should be frozen.

2. The temperature in the kit parcel – the cooling packs should still be cold.3. Transfer kit box to a refrigerator at approximately +4-8˚C immediately for storage.4. Transfer samples to freezer (preferably –80˚C) immediately for storage.5. Please report the above when returning the Report Sheet (see Study Protocol).6. Please confirm the arrival of the kits by reporting to [email protected]

The shipment contains one custom assembled fathead minnow (FHM) Vtg ELISA kit(V01018402 Pilot kit). For convenience, we have prepared two packs of two ELISA plateseach. There are also two vials of Dilution buffer concentrate and TMB substrate, and two vialsof Vtg standard. Half of the components are to be used on day one, the other half on day two.Note that only one vial of Detecting antibody (vial E) is included, this must be u sed on bothassay days.

A Study Protocol is sent as a s eparate file. This contains an o verview of enclosed samples, astudy protocol with plate layouts and Report Sheets. Please return Report and Result sheetselectronically to [email protected].

Again thank you for your interest to participate in the validation, and good luck with theanalysis!

All the bestJanne K. Eidem

Janne K. EidemScientist, R&DBiosense Laboratories ASThormøhlensgate 55NO-5008 Bergen, NorwayTel: +47 55 54 39 80/66Mob: +47 91 81 77 81Fax: +47 55 54 37 71 ***


C: STUDY PROTOCOL AND REPORT SHEET

***


***


D: REPORT SHEET (EXCEL)

Result sheet day 1

Instructions1. Paste values into each plate (Edit - Paste special - Values)2. Remove clear outliers (if %CV > 10)3. Adjust standard curves4. Use equation from each standard curve to calculate Vtg concentrations in samples

Day 1 Plate 21 2 3 4 5 6 7 8 9 10 11 12

A 0.055 0.060 0.066 0.081 0.104 0.176 0.294 0.542 0.994 1.831 3.445 out 6.000B 0.055 0.060 0.064 0.080 0.107 0.170 0.281 0.529 0.941 1.833 3.283 4.219C 0.055 0.060 0.065 0.078 0.107 0.163 0.273 0.494 0.933 1.856 3.294 4.134D 0.055 0.054 0.057 0.053 0.052 0.052 0.053 0.053 0.053 0.054 0.054 0.054E 0.103 0.104 0.101 0.102 0.099 0.105 0.106 0.101 0.100 0.053 0.056 0.055F 0.324 0.322 0.332 0.316 0.302 0.315 0.301 0.318 0.321 0.057 0.053 0.059G 1.588 1.613 1.575 1.551 1.543 1.562 1.609 1.596 1.565 0.053 0.054 0.058H 0.112 0.128 0.097 0.130 0.043 0.122 0.042 0.045 0.039 0.126 0.044 0.044

Plate 21 2 3 4 5 6 7 8 9 10 11 12

A 0.059 0.073 0.073 0.090 0.112 0.171 0.286 0.529 0.943 1.865 3.446 6.000B 0.064 0.069 0.080 0.087 0.115 0.169 0.268 0.496 0.920 1.851 3.320 6.000C 0.064 0.067 0.069 0.079 0.102 0.154 0.261 0.477 0.891 1.758 3.267 6.000D 1.160 1.158 1.141 1.056 1.052 1.013 1.067 1.089 1.051 0.056 0.055 0.056E 0.285 0.274 0.270 0.265 0.263 0.254 0.260 0.263 0.265 0.054 0.058 0.054F 0.100 0.104 0.106 0.107 0.099 0.097 0.097 0.101 0.101 0.060 0.058 0.054G 0.058 0.055 0.062 0.062 0.063 0.063 0.059 0.061 0.063 0.063 0.062 0.058H 0.117 0.098 0.102 0.097 0.098 0.098 0.046 0.043 0.045 0.046 0.044 0.044

STANDARD CURVE PLATE 1

Concentration (ng/ml) NSB 0.05 0.10 0.20 0.39 0.78 1.56 3.13 6.25 12.50 25.00 50.00Absorbance 450 nm 0.055 0.060 0.065 0.080 0.106 0.170 0.283 0.522 0.956 1.840 3.341 4.177%CV (A450nm) 0.0 0.0 1.5 1.9 1.6 3.8 3.7 4.8 3.5 0.8 2.7 1.4NSB-corrected absorbance 0.005 0.010 0.025 0.051 0.115 0.228 0.467 0.901 1.785 3.286 4.122


Concentration (ng/ml) NSB 0.05 0.10 0.20 0.39 0.78 1.56 3.13 6.25 12.50 25.00 50.00Absorbance 450 nm 0.06233 0.07 0.074 0.08533 0.10967 0.16467 0.27167 0.50067 0.918 1.82467 3.34433 6%CV (A450nm) 4.6 4.4 7.5 6.7 6.2 5.6 4.7 5.3 2.8 3.2 2.7 0.0NSB-corrected absorbance 0.007 0.012 0.023 0.047 0.102 0.209 0.438 0.856 1.762 3.282 5.938

SAMPLES PLATE 1Sample ID PJ PQ PB PS WJ WQ WB WS PM PM PM PA PA PA PR PR PRDilution factor 10 10 10 10 10 10 10 10 50 5000 500000 50 5000 500000 50 5000 500000Absorbance 450 nm 0.055 0.052 0.053 0.054 0.103 0.102 0.102 0.055 0.326 1.592 0.112 0.311 1.552 0.098 0.313 1.590 0.042%CV (A450nm) 2.8 1.1 0.0 0.0 1.5 2.9 3.1 2.8 1.6 1.2 13.8 2.5 0.6 48.9 3.4 1.4 7.1NSB-corrected absorbance 0.000 -0.003 -0.002 -0.001 0.048 0.047 0.047 0.000 0.271 1.537 0.057 0.256 1.497 0.043 0.258 1.535 -0.013Calculated concentration 0.002 #NUM! #NUM! #NUM! 0.346 0.341 0.344 #NUM! 2.103 12.742 0.419 1.983 12.398 0.314 2.001 12.725 #NUM!Corrected for dilution factor 0.02 #NUM! #NUM! #NUM! 3.46 3.41 3.44 #NUM! 105.17 63711.49 ###### 99.13 61991 ###### 100.07 63625.44 #NUM!

SAMPLES PLATE 2Sample ID BJ BQ BB BS WM WM WM WA WA WA WR WR WRDilution factor 10 10 10 10 100 10000 1000000 100 10000 1000000 100 10000 1000000Absorbance 450 nm 1.153 1.040 1.069 0.056 0.276 0.103 0.058 0.261 0.101 0.063 0.263 0.100 0.061%CV (A450nm) 0.9 2.3 1.8 1.0 2.8 3.0 6.0 2.2 5.2 0.9 1.0 2.3 3.3NSB-corrected absorbance 1.091 0.978 1.007 -0.007 0.214 0.041 -0.004 0.198 0.039 0.000 0.200 0.037 -0.001Calculated concentration 9.06 8.10 8.34 #NUM! 1.69 0.31 #NUM! 1.57 0.29 0.00 1.58 0.28 #NUM!Corrected for dilution factor 90.57 80.95 83.40 #NUM! 169.34 3089.53 #NUM! 156.59 2908.65 2178.34 158.22 2805.44 #NUM!

Standard curves

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml Vtg

A4

50

-NS

B



Adjusted standard curves y = 0.1324x1.038

R2 = 0.9977

y = 0.1283x1.0296

R2 = 0.9996

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml Vtg

A4

50

-NS

B



Power (STANDARD CURVE PLATE 1)


***


E: INDIVIDUAL RAW DATALAB 1

Standard curves Day 1 and 2:Adjusted standard curves y = 0.1443x1.0271

R2 = 0.9986

y = 0.1354x1.0401

R2 = 0.9978

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml Vtg

A4

50

-NS

B






R2 = 0.9984

y = 0.1344x1.0253

R2 = 0.9985

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml Vtg

A4

50

-NS

B





Day 1 Plate 11 2 3 4 5 6 7 8 9 10 11 12

A 0.052 0.056 0.063 0.081 0.111 0.176 0.300 0.579 1.097 2.065 3.593 6.000B 0.052 0.057 0.068 0.079 0.107 0.170 0.284 0.553 1.033 2.016 3.429 6.000C 0.054 0.056 0.063 0.078 0.107 0.167 0.287 0.543 0.993 1.930 3.455 4.486D 0.052 0.051 0.051 0.108 0.104 0.105 0.358 0.349 0.349 1.596 1.568 1.618E 0.051 0.052 0.051 0.098 0.096 0.096 0.273 0.276 0.270 1.195 1.230 1.274F 0.059 0.066 0.065 0.095 0.088 0.088 6.000 4.337 4.613 0.052 0.055 0.056G 0.051 0.054 0.056 0.053 0.051 0.051 4.106 3.905 4.023 0.056 0.055 0.052H 0.053 0.054 0.052 0.050 0.051 0.048 0.120 0.119 0.118 0.054 0.052 0.053

Plate 21 2 3 4 5 6 7 8 9 10 11 12

A 0.052 0.058 0.063 0.078 out 0.125 0.174 0.297 0.556 1.034 2.002 3.555 6.000B 0.053 0.059 0.065 0.081 0.107 0.166 0.276 0.515 0.985 1.911 3.452 4.860C 0.053 0.056 0.060 0.079 0.102 0.163 0.263 0.492 0.953 1.820 3.389 4.258D 0.059 0.049 0.049 0.103 0.102 0.102 0.339 0.334 0.338 1.562 1.620 1.655E 2.637 2.565 2.503 4.070 4.783 4.519 4.560 5.560 5.083 0.054 0.055 0.057F 0.081 0.081 0.078 1.198 1.135 1.152 4.606 4.519 4.481 0.052 0.054 0.053G 0.050 0.050 0.050 0.063 0.062 0.062 1.246 1.195 1.195 0.052 0.053 0.053H 0.050 0.043 0.042 0.044 0.042 0.041 0.043 0.042 0.042 0.044 0.041 0.041

Day 2 Plate 11 2 3 4 5 6 7 8 9 10 11 12

A 0.056 0.061 0.067 0.083 0.114 0.175 0.309 0.568 1.043 1.955 3.455 4.574B 0.055 0.058 0.068 0.078 0.109 0.168 0.290 0.559 1.036 1.968 3.514 4.398C 0.052 0.058 0.063 0.078 0.105 0.163 0.295 0.531 1.009 1.894 3.390 4.796D 0.054 0.052 0.052 0.107 0.107 0.109 0.334 0.342 0.344 1.593 1.627 1.714E 0.062 0.055 0.053 0.094 0.088 0.087 0.252 0.252 0.258 1.195 1.162 1.232F 3.096 3.044 2.955 6.000 4.801 4.217 6.000 4.431 4.430 1.552 1.572 1.546G 0.098 0.091 0.088 1.342 1.339 1.322 4.624 6.000 4.462 0.350 0.345 0.363H 0.057 0.057 0.059 0.073 0.074 0.069 1.538 1.538 1.523 0.113 0.115 0.113

Plate 21 2 3 4 5 6 7 8 9 10 11 12

A 0.053 0.061 0.066 0.087 0.115 0.170 0.298 0.519 0.967 1.823 3.635 6.000B 0.054 0.055 0.064 0.079 0.108 0.163 0.279 0.501 0.943 1.829 3.368 6.000C 0.059 0.061 0.068 0.077 0.109 0.158 0.277 0.498 0.927 1.710 3.274 6.000D 0.051 0.050 out 0.061 0.100 0.114 0.098 0.313 0.313 0.317 1.578 1.642 1.603E 0.056 0.054 0.054 0.087 0.083 0.083 5.264 4.565 4.611 0.380 0.388 0.396F 0.052 0.050 0.052 0.052 0.054 0.054 4.264 4.335 4.286 0.121 0.123 0.125G 0.051 0.049 0.051 0.050 0.050 0.050 0.125 0.123 0.123 0.064 0.072 0.068H 0.052 0.052 0.054 0.050 0.054 0.050 0.054 0.055 0.056 0.057 0.055 0.055

***


LAB 2


R2 = 0.9992

y = 0.2094x1.0206

R2 = 0.9993

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml Vtg

A4

50

-NS

B






R2 = 0.9988

y = 0.2218x0.9439

R2 = 0.9986

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml Vtg

A4

50

-NS

B





Day 1 Plate 11 2 3 4 5 6 7 8 9 10 11 12

A 0.090 0.116 0.127 0.150 0.201 0.272 0.424 0.686 1.290 2.369 4.617 7.531B 0.092 0.145 0.117 0.133 0.177 0.254 0.399 0.647 1.235 2.279 4.473 7.581C 0.093 0.112 0.112 0.136 0.181 0.236 0.385 0.647 1.176 2.206 4.317 7.601D 0.093 0.104 0.089 0.165 0.173 0.150 0.378 0.380 0.373 1.607 1.602 1.615E 0.095 0.093 0.092 0.138 0.152 0.139 0.354 0.341 0.344 1.395 1.443 1.454F 4.648 4.378 4.334 7.531 7.521 7.541 7.601 7.531 7.452G 0.136 0.140 0.134 1.507 1.561 1.548 7.433 7.452 7.511H 0.105 0.108 0.089 0.110 0.116 0.110 1.608 1.566 1.506

Plate 21 2 3 4 5 6 7 8 9 10 11 12

A 0.109 0.105 0.111 0.143 0.189 0.296 0.473 0.821 1.550 2.850 5.743 7.511B 0.090 0.103 0.114 0.138 0.181 0.276 0.455 0.785 1.489 2.733 5.396 7.601C 0.126 0.100 0.111 0.145 0.182 0.264 0.430 0.743 1.430 2.652 5.614 7.591D 0.090 0.088 0.089 0.161 0.160 0.158 0.475 0.480 0.494 2.350 2.385 2.456E 0.097 0.112 0.103 0.201 0.148 0.169 7.683 7.672 7.632F 0.306 0.317 1.252 0.092 0.091 0.091 7.395 7.462 7.414G 0.094 0.098 0.103 0.096 0.098 0.091 0.204 0.203 0.347H

Day 2 Plate 11 2 3 4 5 6 7 8 9 10 11 12

A 0.080 0.316 0.130 0.139 0.193 0.276 0.454 0.874 1.597 3.043 5.478 7.906B 0.303 0.195 0.111 0.997 0.180 0.259 0.446 0.802 1.427 2.707 5.111 7.652C 0.075 0.090 0.119 0.811 0.177 0.249 0.442 0.776 1.420 2.693 4.935 7.704D 0.104 0.131 0.093 0.161 0.163 0.152 0.454 0.469 0.472 2.259 2.255 2.422E 0.101 0.223 0.095 0.162 0.148 0.149 0.386 0.373 0.381 1.836 1.898 1.745F 3.910 3.761 3.935 8.583 7.109 6.962 8.481 7.611 9.175G 0.185 0.263 0.129 1.773 1.738 1.700 7.309 6.639 6.495H 0.117 0.176 0.251 0.117 0.112 0.115 2.050 1.926 1.973

Plate 21 2 3 4 5 6 7 8 9 10 11 12

A 0.093 0.105 0.142 0.153 0.190 0.285 0.445 0.797 1.401 2.819 4.716 7.511B 0.128 0.110 0.116 0.135 0.180 0.283 0.421 0.728 1.299 2.571 4.453 7.611C 0.091 0.125 0.112 0.145 0.176 0.221 0.400 0.699 1.247 2.357 4.424 7.601D 0.091 0.096 0.101 0.181 0.169 0.169 0.482 0.497 0.472 2.258 2.253 2.265E 0.089 0.090 0.093 0.125 0.124 0.125 7.873 7.190 7.581F 0.105 0.101 0.093 0.092 0.102 0.095 7.501 7.423 7.414G 0.092 0.106 0.110 0.093 0.098 0.095 0.258 0.255 0.305H

***


LAB 3


R2 = 0.9997

y = 0.0553x0.9793

R2 = 0.999

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml Vtg

A4

50

-NS

B






R2 = 0.9989

y = 0.0704x0.9712

R2 = 0.9971

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml Vtg

A4

50

-NS

B





Day 1 Plate 11 2 3 4 5 6 7 8 9 10 11 12

A 0.060 0.060 0.063 0.067 0.077 0.098 0.137 0.217 0.370 0.667 1.253 2.271B 0.061 0.060 0.064 0.069 0.077 0.097 0.131 0.209 0.347 0.636 1.180 2.179C 0.061 0.063 0.065 0.071 0.080 0.094 0.132 0.204 0.343 0.631 1.177 2.197D 0.059 0.059 0.060 0.080 0.077 0.076 0.157 0.158 0.161 0.632 0.632 0.694E 0.059 0.059 0.061 0.075 0.073 0.070 0.107 0.145 0.105 0.335 0.342 0.346F 1.591 1.461 1.428 4.000 4.000 4.000 4.000 4.000 4.000 0.061 0.062 0.061G 0.076 0.133 0.077 0.584 0.590 0.577 4.000 4.000 4.000 0.061 0.062 0.062H 0.061 0.061 0.062 0.067 0.066 0.069 0.538 0.535 0.555 0.062 0.060 0.062

Plate 21 2 3 4 5 6 7 8 9 10 11 12

A 0.064 0.066 0.068 0.076 0.084 0.107 0.151 0.248 0.426 0.773 1.321 2.396B 0.059 0.063 0.064 0.070 0.083 0.104 0.150 0.233 0.418 0.732 1.345 2.277C 0.066 0.067 0.075 0.086 0.109 0.150 0.259 0.415 0.691 1.388 2.435D 0.064 0.066 0.063 0.089 0.087 0.088 0.179 0.180 0.185 0.717 0.715 0.666E 0.064 0.062 0.064 0.066 0.072 0.069 4.000 4.000 4.000 0.065 0.064 0.074F 0.065 0.065 0.068 0.069 0.067 0.069 2.655 2.536 2.532 0.066 0.071 0.069G 0.078 0.078 0.084 0.080 0.084 0.084 0.097 0.097 0.099 0.067 0.072 0.072H 0.069 0.072 0.069 0.073 0.070 0.072 0.067 0.075 0.070 0.067 0.071 0.074

Day 2 Plate 11 2 3 4 5 6 7 8 9 10 11 12

A 0.058 0.056 0.057 0.062 0.075 0.097 0.140 0.230 0.386 0.759 1.380 2.501B 0.053 0.053 0.057 0.061 0.072 0.093 0.135 0.209 0.345 0.710 1.259 2.383C 0.056 0.056 0.057 0.062 0.073 0.094 0.138 0.211 0.368 0.699 1.253 2.391D 0.060 0.062 0.056 0.074 0.075 0.078 0.161 0.160 0.160 0.654 0.667 0.718E 0.054 0.053 0.059 0.065 0.074 0.065 0.113 0.122 0.109 0.374 0.377 0.425F 1.350 1.191 1.222 4.000 4.000 4.000 4.000 4.000 4.000 0.058 0.056 0.057G 0.072 0.071 0.068 0.605 0.616 0.639 4.000 4.000 4.000 0.054 0.054 0.056H 0.067 0.058 0.056 0.064 0.071 0.067 0.574 0.566 0.562 0.058 0.057 0.056

Plate 21 2 3 4 5 6 7 8 9 10 11 12

A 0.069 0.072 0.079 0.094 0.133 0.181 0.294 0.478 0.999 1.734B 0.058 0.059 0.067 0.074 0.083 0.116 0.189 0.258 0.494 0.950 1.598 2.640C 0.062 0.064 0.067 0.076 0.087 0.124 0.176 0.290 0.482 0.937 1.862 2.797D 0.060 0.061 0.061 0.089 0.090 0.092 0.211 0.191 0.188 0.771 0.864 0.885E 0.061 0.073 0.068 0.068 0.066 0.070 4.000 4.000 4.000 0.069 0.061 0.061F 0.071 0.075 0.075 0.069 0.074 0.073 2.321 2.783 2.615 0.076 0.066 0.070G 0.073 0.077 0.083 0.067 0.071 0.067 0.098 0.109 0.103 0.082 0.074 0.079H 0.087 0.083 0.098 0.083 0.087 0.079 0.087 0.088 0.085 0.082 0.075 0.084

***


LAB 4

Standard curves Day 1 and 2:Adjusted standard curves

y = 0.0376x1.0542

R2 = 0.9936

y = 0.0646x0.8492

R2 = 0.9935

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml Vtg

A4

50

-NS

B






R2 = 0.9814

y = 0.2315x0.8386

R2 = 0.9978

0.001

0.01

0.1

1

10

0.01 0.1 1 10 100

ng/ml Vtg

A4

50

-NS

B





Day 1 Plate 11 2 3 4 5 6 7 8 9 10 11 12

A 0.068 0.548 0.055 0.223 0.059 0.074 0.107 0.166 0.366 0.546 1.173 1.493B 0.049 0.046 0.049 0.052 0.061 0.196 0.147 0.280 0.352 0.516 0.934 2.003C 0.046 0.048 0.345 0.052 0.060 0.088 0.122 0.180 0.361 0.483 1.070 1.745D 0.044 0.057 0.046 0.064 0.067 0.055 0.083 0.126 0.107 0.407 0.370 0.389E 0.087 0.082 0.086 0.060 0.057 0.090 0.112 0.112 0.204 0.357 0.223 0.327F 0.477 0.401 0.649 3.550 3.439 3.551 3.691 3.731 3.659 G 0.060 0.051 0.068 0.454 0.447 0.472 4.000 4.000 4.000 H 0.164 0.055 0.105 0.108 0.078 0.070 0.374 0.310 0.383 0.056 0.053 0.073

Plate 21 2 3 4 5 6 7 8 9 10 11 12

A 0.146 0.075 0.104 0.102 0.135 0.155 0.161 0.209 0.457 0.594 1.212 1.900B 0.137 0.150 0.183 0.132 0.165 0.361 0.384 0.349 0.404 0.674 1.082 1.736C 0.302 0.150 0.119 0.090 0.171 0.192 0.399 0.363 0.444 0.696 1.128 2.026D 0.155 0.088 0.106 0.146 0.092 0.071 0.214 0.176 0.140 0.440 0.375 0.394E 1.143 1.807 0.266 0.203 0.269 0.225 3.543 3.702 3.576 F 0.139 2.836 0.264 1.453 0.216 0.231 1.538 1.793 1.132 G 0.263 2.283 2.297 0.101 1.420 0.186 1.133 0.113 0.078 0.203 0.116 0.065H

Day 2 Plate 11 2 3 4 5 6 7 8 9 10 11 12

A 0.447 0.393 0.200 0.309 0.240 0.472 0.569 1.628 1.935 2.677 3.800 4.000B 0.381 0.330 0.214 0.313 0.288 0.524 0.705 0.940 1.620 2.626 3.740 4.000C 0.382 0.327 0.220 0.183 0.265 0.604 0.637 1.373 1.622 2.510 3.709 4.000D 0.168 0.152 0.167 0.245 0.364 0.307 0.746 0.614 0.596 2.570 2.380 2.216E 0.206 0.203 0.366 0.240 0.268 1.087 0.414 0.483 1.825 1.434 1.514 1.394F 3.660 3.678 3.683 3.952 3.944 3.968 4.000 4.000 4.000 G 0.291 0.244 0.223 2.245 2.247 2.194 4.000 4.000 4.000 H 0.214 2.283 0.279 3.900 0.250 0.245 2.805 2.745 2.679 0.157 0.198 0.368

Plate 21 2 3 4 5 6 7 8 9 10 11 12

A 2.064 0.287 0.139 0.173 0.242 0.361 0.440 0.763 1.108 2.101 3.582 4.000B 0.235 0.080 0.913 0.224 0.230 0.536 0.424 0.721 1.221 2.238 3.506 4.000C 0.927 0.122 3.789 1.950 0.215 0.791 0.408 0.674 1.189 2.213 3.445 4.000D 0.087 0.190 0.072 0.160 0.158 0.157 3.699 2.045 0.451 1.973 1.790 1.790E 0.095 0.133 0.137 0.105 0.090 0.088 4.000 4.000 4.000 F 0.074 0.097 0.069 0.065 0.092 0.074 4.000 4.000 4.000 G 0.155 0.071 0.065 0.139 0.104 0.059 0.230 0.224 1.087 0.091 0.231 0.144H

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inter-laboratory validation of a homologous elisa to quantify vitellogenin … · 2020. 3. 6. ·...

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