Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Inter-Laboratory Validation of a homologous
ELISA to quantify Vitellogenin in the
fathead minnow (Pimephales promelas)
Janne K. Eidem, Hans Kleivdal and Anders Goksøyr
Biosense Laboratories AS
Thormøhlensgt 55
N-5008 Bergen
http://www.biosense.com
Contact:
Janne K. Eidem
+47 55543980
janne@biosense.com
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
TABLE OF CONTENTS
SUMMARY
INTRODUCTION
The fathead minnow Vitellogenin ELISA
Brief summary of method
PURPOSE OF THE VALIDATION
AIMS OF THE VALIDATION STUDY
STUDY DESIGN
Participant Laboratories
Sample preparation and spiking
Sample and test kit shipments
Data reporting and handling
VALIDATION RESULTS
Applicability (Scope)
Precision
Between-Day Precision RSDr
Between-Laboratory Precision RSDR
Accuracy
DISCUSSION
CONCLUSION
ACKNOWLEDGEMENTS
REFERENCES AND LINKS
APPENDIX
A. Single-Lab Validation Report
B. Letter to participants
C. Study protocol and report sheet
D. Result sheet
E. Individual raw data
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
SUMMARY
Vitellogenin (Vtg) is an established and sensitive endpoint for analysis of endocrine
disruption in fish. The widespread use of Vtg in this regard has led to the need for
standardised assays to quantify Vtg in fish samples. Fathead minnow (Pimephales
promelas) is an important fish test species used in ecotoxicology laboratories across
the world.
Based on monoclonal antibodies raised against fathead minnow (FHM) Vtg, we
have 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 was
carried out in order to determine the performance of the FHM Vtg ELISA in four
different laboratories. Study samples consisted of both plasma, whole body
homogenate (WBH) samples and ELISA kit Dilution buffer. The results are
summarised in Table 1, and show that the FHM Vtg ELISA is suitable for
quantification of Vtg in both plasma and WBH samples from FHM in different
laboratories.
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
16.4
Between-Lab Reproducibility Precision, RSDR
>50
18.6
Recovery
50-200
69.4
Bias
-30.6
1
Goksøyr et al 2003
***
INTRODUCTION
Vitellogenin (Vtg) is a large phospholipoglycoprotein, which functions as the egg yolk
precursor in oviparous vertebrates such as fish. The Vtg protein is produced in the
liver and secreted from the liver cells through the secretory pathway, enters the
blood 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 (for
reviews, see Mommsen & Walsh, 1988; Arukwe & Goksøyr, 2003). More than a
decade ago, several studies demonstrated that also male fish, caught in rivers and
streams, 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. Since
then, numerous studies have shown the fish Vtg to be a highly responsive
biomarker for estrogenic compounds in both in vitro hepatocyte cell cultures, in vivo
aquaria studies, and field studies (for reviews, see Kime, 1995; Sumpter & Jobling,
1995; Arukwe & Goksøyr, 1998; 2003). Vtg induction in fish has now become an
accepted measure of xenoestrogenic potency of chemicals, effluents, and
discharges.
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
X
Liver
Vitellogenin
Zona radiata P.
Gonads
Estradiol
Figure 1: In response to oestradiol or xenoestrogens (x), Vitellogenin (egg yolk precursor) and zona
radiata proteins (egg shell precursors) are produced in the liver and transported via the blood to the
gonads.
Within international bodies such as the Organization for Economic Cooperation and
Development (OECD), work is ongoing to develop screening and testing
programmes for endocrine disrupting effects of new chemicals. In the focus of this
development are small fish test species including the fathead minnow (Pimephales
promelas), zebrafish (Danio rerio), and Japanese medaka (Oryzias latipes). These
fish share several attributes that make them ideal test species for reproductive
toxicity 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 methods
for measuring Vtg levels in these fish species, assays that are readily available and
give reproducible results in different laboratories. The enzyme-linked
immunosorbent assay (ELISA) is a sensitive laboratory technique widely used to
detect and quantify antigens in a variety of biological samples. They can be
quantitative (with a standard curve) or qualitative (semi-quantitative - without a
standard curve). The two most widely used principles for quantitative detection of
proteins are the competitive ELISA and the sandwich ELISA techniques (Crowther,
2001).
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
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 FHM
Vtg in the standard or sample added to the ELISA well (Figure 2). After unbound
components are washed away, a Detecting Ab, labelled with the enzyme
horseradish peroxidase (HRP), is added. This Ab binds to a different part of the Vtg
molecule, creating a sandwich of antibodies and Vtg. Addition of the HRP substrate
Tetramethyl Benzidine (TMB) results in a colour reaction where the enzyme
catalyses the conversion of this uncoloured substrate to a blue product. After
development, the reaction is stopped by addition of a mild sulphuric acid, changing
the colour from blue to yellow. The colour intensity is measured using a microplate
reader with a 450 nm filter, and is proportional to the concentration of Vtg in the
standard/sample.
All absorbance levels are corrected for non-specific background reading (NSB), and
a calibration curve is created by plotting Vtg concentration on the x-axis and the
corresponding absorbance level on the y-axis (Figure 3). A 4 parameter or a log-log
curve fit can be used to describe the relationship between the concentration and
the signal. The equation for this calibration curve is then used to calculate the Vtg
concentration in plasma or WBH samples.
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
FHM Vtg standard
100
10
90
80
70
60
50
0.1
40
%CV
A450-NSB
1
30
0.01
20
10
0.001
0.01
0
0.1
1
10
100
ng/ml
Standard
Within-Day Precision
Between-Day Precision
Figure 3: Fathead minnow Vtg standard curve, compiled from 15 assays performed during the
Single-Laboratory Validation (using Microsoft Excel). The secondary y-axis shows the Within-Day and
Between-Day Repeatability Precisions (RSDr) for the standard curves.
A Single-Laboratory Validation of the FHM Vtg ELISA was performed at Biosense
Laboratories AS (Eidem et al 2005a). The validation demonstrated that the ELISA is
both sensitive, precise and reliable and thus fit for its intended purpose. Figure 3
shows a representative standard curve derived from 15 assays performed during
the Single-Lab Validation, including Between-Day and Within-Day Repeatibility
Precision (RSDr) for the whole standard curve concentration range. Table 2
summarises the results of the Single-Laboratory Validation. The complete results
from this validation are presented in Appendix A.
Table 2: Summary of Single-Laboratory Validation results (Eidem et al 2005a)
Performance characteristics
Aim1
Value
Selectivity
Matrix blank < LOD
No response at minimum
(with the necessary dilution dilution = 1:50 (plasma), 1:100
factor to avoid matrix effects)
(WBH)
Calibration
Standard curve working range Standard curve working range
>10-fold, preferably 50-100 0.1-25 ng/ml (250-fold)
fold to be practical with the
dynamic range 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
200 – 500 ng/ml
4.68 ng/ml (plasma, 1:50)
(=LOQ x necessary matrix dilution)
11.35 ng/ml (WBH, 1:100)
Comparison with Biosense
R2>0.99
Carp Vtg ELISA kit
Comparison with competitor
R2>0.99
FHM Vtg ELISA kit
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
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 can
also 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 and
screening, various studies have been conducted over the recent years, involving
both non-commercial laboratory methods and a few commercially available ELISA
kits
(for
zebrafish
Vtg
inter-comparison
study
see
http://abstracts.co.allenpress.com/pweb/setac2003/document/?ID=30012, for an
overview
of
validation
status
in
the
US,
see
http://www.epa.gov/scipoly/oscpendo/assayvalidation/status.htm). These studies
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
have demonstrated the variance between different Vtg standards, between different
assays and between different laboratories using the same assay.
Clearly, there is a need for standardised, reproducible methods/kits that have been
thoroughly characterised, with validation data describing the assay’s performance,
thus giving the end user reasonable expectancies. The aim of the following study
was to obtain inter-laboratory data on Precision and Accuracy, according to
international guidelines (http://www.aoac.org).
***
AIMS OF THE VALIDATION STUDY
In a 2003 document, Biosense took the initiative to stress the need for standardised
validations of Vtg standards and assays (Goksøyr et al, 2003). In this document,
which was based on experience from participation in studies comparing Vtg
methods, a set of performance criteria was suggested, to be met by Vtg
quantification methods (see under “Aims” in Table 1).
In this Inter-Laboratory Validation, a set of characteristics was analysed, chosen on
the basis of international guidelines from the AOAC (http://www.aoac.org). The
terminology involved in validation work is often confusing, and depends largely on
which set of guidelines one looks at. Definitions used in this report is based on
http://www.aoac.org/intaffairs/analytical_terminology.htm
This study aimed to obtain Inter-Laboratory Validaion data on the following
parameters:
1. Applicability (Scope)
The analytes, matrices and concentrations for which a method of analysis may be
used satisfactorily
2.
Precision (Within-Laboratory, Between-Laboratory)
Closeness of agreement between independent test results obtained under
stipulated 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 to
participate in the validation. These laboratories have long, previous experience with
the ELISA technique, removing the lack of experience factor from the results.
***
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Biosense Laboratories AS
Inter-Laboratory validation 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 having
non-detectable levels of Vtg at a 1:100 dilution were pooled.
WBH from male fathead minnows was a gift from Robert Bringolf, Iowa State
University, 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 of
purified, non-lyophilised Vtg. Samples were mixed thoroughly, aliquoted into suitable
volumes and frozen at –80oC. Concentrations in spiked samples were 5, 25 and
125 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 WBH
with low and medium levels of Vtg was a gift from Robert Bringolf, Iowa State
University, USA. WBH from E2-treated FHM was prepared at Biosense
Laboratories. Samples were mixed thoroughly, aliquoted into suitable volumes and
frozen 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 and
that 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 4oC
upon arrival.
***
DATA REPORTING AND HANDLING
Each participating laboratory was given detailed instructions, plate layout and a
report 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 at
Biosense Laboratories AS):
1. Standard curves were adjusted according to kit protocol.
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
2. Each standard and sample dilution had been performed in triplicates. If the CV
between 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 standard
curve working range were discarded.
4. If more than one sample dilution fell within the working range of the standard
curve, the average of these was calculated. If the results from these different
dilutions had a CV>50%, this data point was excluded.
***
VALIDATION RESULTS
APPLICABILITY (SCOPE)
Applicability (Scope): The analytes, matrices and concentrations for which a method
may be used satisfactorily.
This Inter-Laboratory Validation was performed in order to validate the performance
of the FHM Vtg ELISA, developed for use in endocrine disruptor screening tests.
The same material was analysed by four different laboratories with previous ELISA
skills. 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 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 (0.1-25 ng/ml). The following
validation data demonstrate that the FHM Vtg ELISA kit is suitable for Vtg analysis in
the matrices and in the concentrations ranges included in the validation.
***
PRECISION
Precision is the closeness of agreement between test results obtained under stipulated
conditions.
• Repeatability Precision (same laboratory and operator, samples, equipment, short
time intervals), separated into Within-Day and Between-Day Repeatability
precision, usually expressed as relative standard deviation, RSDr.
• Reproducibility Precision (different laboratories, equipment and operators, same
samples). Usually expressed as relative standard deviation, RSDR.
Spiked and unspiked plasma and WBH sample blanks, as well as the ELISA kit
Dilution buffer, were analysed. On two different days, one aliquot was thawed,
diluted 1:10 and analysed in triplicates. Unspiked plasma and WBH samples were
analysed at three different dilutions according to kit protocol (plasma 1:50, 1:5000
and 1:500000; WBH 1:100, 1:10000 and 1:1000000).
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Table 3 shows the compiled data from all four laboratories and both days as well as
Between-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 laboratory
Overall RSDr: Average RSDr for all samples and laboratories
Table 3: Between-Day Repeatability Precision RSDr Results from four laboratories, showing
measured Vtg levels (ng/ml) and RSDr (%).
Lab1
code
Day 1
Lab2
Day 2
Average RSD Day 1
PJ
<w.r.
<w.r.
-
PQ
0.4
0.4
PB
2.0
PS
WJ
r
Lab3
Day 2
Average RSD Day 1
-
<w.r.
<w.r.
-
0.4
2.1
0.3
0.3
2.0
2.0
1.9
1.4
10.0
10.5
10.3
3.1
<w.r.
<w.r.
-
-
WQ
0.3
0.3
0.3
WB
1.5
1.4
WS
7.7
7.6
BJ
<w.r.
BQ
0.4
BB
r
Lab4
Day 2
Average RSD Day 1
-
<w.r.
<w.r.
-
0.3
1.8
0.4
0.4
1.6
1.5
10.0 2.1
8.5
10.4
9.4
<w.r.
<w.r.
-
13.3 0.2
0.3
1.5
5.4
1.2
7.7
1.3
7.4
<w.r.
-
-
0.4
0.4
1.9
2.0
1.9
2.0
BS
10.5
10.9
PM
952
1180
PA
38642
54454
PR
3940983 4883251 4412117
WM
7.92
<w.r.
WA
27.0
WR
469214
r
Day 2 Average RSD
r
-
<w.r.
<w.r.
-
0.4
6.4
0.5
nd
0.5
-
2.1
2.1
0.8
nd
1.6
1.6
-
14.5 13.0
12.5
12.7
2.8
8.1
10.1
9.1
15.7
-
<w.r.
<w.r.
-
-
1.0
<w.r.
1.0
-
0.2
17.7 <w.r.
<w.r.
-
-
<w.r.
0.2
0.2
-
1.2
1.2
0.1
1.0
1.2
1.1
13.6 1.7
nd
1.7
-
8.0
7.7
5.4
6.1
6.7
6.4
6.8
7.1
5.5
6.3
17.9
<w.r.
<w.r.
-
-
<w.r.
<w.r.
-
-
<w.r.
<w.r.
-
-
0.3
0.3
0.3
17.5 0.4
0.4
0.4
11.8 nd
<w.r.
-
-
5.2
1.8
1.8
1.8
1.7
2.2
1.9
2.1
8.2
<w.r.
nd
-
-
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
1066
15.2 1124
919
1021
14.2 1574
1192
1383
19.6 2289
849
1569
64.9
46548
24.0 40192
37687
38939
4.6
57282
56422
56852
1.1
nd
nd
-
-
15.1 4084024 4374596 4229310
4.9
5273682 5116503 5195092
2.1
3935890 >w.r.
3935890
-
7.92
-
<w.r.
<w.r.
-
-
<w.r.
<w.r.
-
-
nd
<w.r.
-
-
22.4
24.7
13.3 26.3
13.1
19.7
47.1 <w.r.
<w.r.
-
-
nd
<w.r.
-
-
516990
493102
6.9
728601
603104
29.4 618288
478045
548166
18.1 nd
Average RSDr
7.9
477607
12.4
409530 409530
7.7
Overall RSDr for all laboratories:
-
-
37.4
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 two
different days varied between 7.7 and 37.4% for the different laboratories. The
overall RSDr for all four laboratories was 16.4%. Note that for Lab 4 RSDr was only
obtainable for four samples.
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Between-Laboratory Reproducibility Precision RSDR:
RSDR: Variation between results obtained by different laboratories for one sample (average
of two days).
Overall RSDR : Average RSDR for all laboratories and samples
Table 4: Between-Laboratory Reproducibility Precision RSDR. Results from four laboratories, showing
measured Vtg levels (ng/ml, average of two days) and RSDR (%)
code
Average day1-2, Average day1-2, Average day1-2, Average day1-2, Average
Lab1
Lab2
Lab3
Lab4
Lab1-4
PJ
PQ
0.4
0.3
0.4
0.5
0.4
PB
2.0
1.5
2.1
1.6
1.8
PS
10.3
9.4
12.7
9.1
10.4
WJ
1.0
WQ 0.3
0.2
0.3
WB 1.5
1.2
1.1
1.7
1.3
WS 7.7
7.7
6.4
6.3
7.0
BJ
BQ
0.4
0.3
0.4
0.4
BB
2.0
1.8
2.1
1.9
BS
10.7
10.8
12.0
8.1
10.4
PM 1066
1021
1383
1569
1260
PA
46548
38939
56852
47446
PR
4412117
4229310
5195092
3935890
4515561
WM 7.9
7.9
WA 24.7
19.7
22.2
WR 493102
603104
548166
409530
528325
Overall Between-Laboratory RSDR
RSD
Lab 1-4
18.5
16.7
16.3
15.0
17.7
12.3
15.6
7.4
21.0
37.6
20.0
12.6
28.7
20.6
18.6
R
“-“ indicates that data was not obtainable for this sample (see Table 3)
Table 4 shows that the variation RSDR between results obtained in four different
laboratories varied between 7.4 and 37.6% for the different samples. The overall
RSDR for all samples was 18.6%.
***
ACCURACY
Accuracy (Trueness) is the closeness of agreement between a test result and an
accepted reference value. Recovery is the proportion of the amount of analyte, present
in or added to, the analytical portion, which is extracted and presented for
measurement. Bias is the difference between the test results and an accepted reference
value.
Recovery and Bias in spiked samples were determined using the following formulas:
Recovery = (C1-C2)/C3 x 100
Where C1= concentration measured in spiked sample, C2= concentration measured in unspiked
sample, C3= theoretical concentration.
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Bias = (C3-(C1-C2))/C3 x 100
Where C1= concentration measured in spiked sample, C2= concentration measured in unspiked
sample, 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)
Average
Lab1-4 (ng/ml)
Recovery
Lab 1-4 (%)
Plasma
0
0.5
2.5
12.5
0
0.5
2.5
12.5
0
0.5
2.5
12.5
0.4
1.8
10.4
1.0
0.3
1.3
7.0
0.4
1.9
10.4
74.8
73.3
83.0
51.7
52.3
56.1
72.7
77.7
83.2
WBH
Buffer
PJ
PQ
PB
PS
WJ
WQ
WB
WS
BJ
BQ
BB
BS
Overall Recovery
Bias
69.4
-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 type
and spike concentration, with an overall Recovery of 69.4%. See Discussion for
more on this. One lab reported a false positive on one day (sample WJ, Lab 4, see
Table 3).
***
DISCUSSION
An Inter-Laboratory Validation was performed to validate the performance of the
Biosense FHM Vtg ELISA in four different laboratories. A set of 18 plasma, WBH
and Buffer samples, comprising both spiked and unspiked samples, was analysed
twice, on two different days. Variation within laboratories (between days) and
between laboratories was analysed, as well as the ability of the assay to measure
correct values in spiked samples.
Kits and samples were successfully received and handled by participants, and data
sets were obtained from all four laboratories.
Samples analysed on two different days determined the Between-Day Repeatability
Precision (RSDr). The RSDr varied from 7.7-37.4% for the different laboratories, with
an overall RSDr of 16.4%. This value was elevated by a high variation and a low
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
number of RSDr values (only from four samples) from Lab 4. If the RSDr values of
this laboratory are excluded, the overall RSDr is reduced to 9.4%.
Samples analysed by different laboratories allowed the determination of BetweenLaboratory Reproducibility Precision (RSDR). The RSDR varied between 7.4 and
37.6% 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 concentrations
corresponding to low, medium and high concentration levels of the standard curve
(working range = 0.1-25 ng/ml, see Single-Laboratory Validation, Appendix A/Eidem
et al, 2005a). The Recovery varied somewhat with sample type and spike level: with
Dilution buffer giving the highest and WBH samples giving the lowest Recovery.
Higher spike concentrations gave somewhat higher Recovery. An overall Recovery
of 69.4% was obtained, averaged over all sample concentrations and types. This
value 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). For
this reason, spiking of samples should ideally be done on the day of analysis to
avoid freeze-thaw cycles and degradation. However, in order to obtain data for
determination of Between-Day and Between-Laboratory Precision, samples had to
be diluted, aliquoted and frozen. This is likely to have affected the Recovery of Vtg in
spiked samples. Spiking/recovery experiments performed in the Single-Laboratory
Validation, without freezing and thawing of the spiked samples, indicate recovery in
both plasma and WBH between 79-106% (see Table 2 and Single-Laboratory
Validation, Appendix A/Eidem et al, 2005a).
Some of the laboratories (especially Lab 4, but also Lab 2) reported high variance
between replicate wells, caused by contamination of wells with enzyme-labelled
Detecting Ab and a resulting unspecific colour development. Generally, one or more
of 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 same
reasons as variation between replicate wells. Variation is reduced by ensuring
consistent techniques when preparing standards, sample and antibody dilutions,
when adding the solutions and when performing washing step. Also, keeping
temperatures and incubation times consistent is important. These factors should be
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
relatively easy to overcome with practise and proper maintenance of pipettes and
equipment.
In addition to variation arising during performance of the assay, some variation is
also inherent in the assay itself. There will be some variation between different
plates, 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 washing
buffer than the one enclosed in the kit had been used – this wash buffer contained
Na-azide as preservant, and which is known to inhibit HRP. On Day 2 the kit wash
buffer 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 accuracy
and that it is fit for its intended purpose, namely reliable quantification of Vtg in
plasma or WBH samples from the fathead minnow (Pimephales promelas).
***
ACKNOWLEDGEMENTS
Biosense Laboratories AS would like to thank the following participants for donating
their time and for their valuable input on the assay (in alphabetical order based on
institution):
Dr. Nancy Denslow and Dr. Kevin J. Kroll
University of Florida, Gainesville, US
Phone: (352)-392-4700
Fax: (352)-392-4707
E-mail: kkroll@ufl.edu
Dr. Grace Panter
AstraZeneca, Brixham Environmental Laboratory
Brixham, UK
Phone: 01803 884261
Fax: 01803882974
E-mail: grace.panter@brixham.astrazeneca.com
Dr. Charles Tyler and Dr. Ronny van Aerle
University of Exeter
Exeter, UK
Phone: 01392 264450
Fax: 01392 263700
E-mail: C.R.Tyler@exeter.ac.uk, R.Van-Aerle@exeter.ac.uk
Page 15
***
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
REFERENCES AND LINKS
AOAC “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 in
fish. Sarsia 83:225-241.
Arukwe A, Goksøyr A (2003). Eggshell and egg yolk proteins in fish: hepatic proteins for the
next generation. Oogenetic, population, and evolutionary implications of endocrine
disruption. Comp Hepatol 2: 4.
Brion F, Nilsen BM, Eidem JK, Goksøyr A, Porcher JM (2002). Development and validation
of an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio
rerio). 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 homologous
ELISA 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 fish
vitellogenin assays as an endpoint in endocrine disruptor testing and screening – a
proposal. http://www.biosense.com/Docs/GoksoyrEtal2003.pdf
Jobling S, Nolan M, Tyler CR, Brighty G, Sumpter JP (1998). Widespread sexual disruption
in wild fish. Environ Sci Technol 32: 2498–2506.
Kime DE (1995). The effects of pollution on reproduction in fish. Rev Fish Biol Fisheries
5:52-96
Mommsen TP, Walsh PJ (1988). Vitellogenesis and oocyte assembly. In: Hoar WS, Randall
VJ (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 species
used in endocrine disruptor screening. Anal Bioanal Chem 378:621
http://www.springerlink.com, DOI 10.1007/s00216-003-2241-2
Norberg B, Haux C (1985). Induction, isolation and a characterization of the lipid content of
plasma 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). Estrogenic
effects of effluents from sewage treatment works. Chem Ecol 8: 275-285.
Silversand C, Haux C (1995). Fatty acid composition of vitellogenin from four teleost
species. J Comp Physiol 164B: 593-599.
Sumpter JP, Jobling S (1995). Vitellogenesis as a biomarker for estrogenic contamination of
the 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
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
APPENDIX
A: SINGLE-LAB VALIDATION REPORT
Single-Laboratory Validation of a homologous ELISA to quantify
Vitellogenin in the
fathead minnow (Pimephales promelas)
Janne K. Eidem, Hans Kleivdal and Anders Goksøyr
Biosense Laboratories AS
Thormøhlensgt 55
N-5008 Bergen
http://www.biosense.com
Contact:
Janne K. Eidem
+47 55543980
janne@biosense.com
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Inter-Laboratory validation FHM Vtg ELISA
TABLE OF CONTENTS
SUMMARY
INTRODUCTION
The fathead minnow Vitellogenin ELISA
Brief summary of method
PURPOSE OF THE VALIDATION
AIMS OF THE VALIDATION STUDY
Definitions
VITELLOGENIN STANDARD
Source for RM purification
Purification of RM
Quality assurance of RM
Quantification of RM
Stabilization of RM
SAMPLE PREPARATION AND SPIKING
VALIDATION RESULTS
Applicability (Scope)
Calibration
LoD and LoQ (sensitivity)
Selectivity (matrix effect)
Repeatability Precision (intra- and inter-assay variation)
Accuracy
Ruggedness
Comparison with existing methods
DISCUSSION
CONCLUSION
REFERENCES AND LINKS
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SUMMARY
Vitellogenin (Vtg) is an established and sensitive endpoint for analysis of endocrine
disruption in fish. The widespread use of Vtg in this regard has led to the need for
standardised assays to quantify Vtg in fish samples. Fathead minnow (Pimephales
promelas) is an important fish test species used in ecotoxicology laboratories across
the world.
Based on monoclonal antibodies raised against fathead minnow (FHM) Vtg, we
have developed a quantitative sandwich enzyme-linked immunosorbent assay
(ELISA) for FHM Vtg. A Single-Laboratory Validation was performed according to
international guidelines. Study samples consisted of both plasma, whole body
homogenate (WBH) and ELISA kit Dilution buffer. The results are summarised in
Table 1, and show that the FHM Vtg ELISA is suitable for quantification of Vtg in
both plasma and WBH samples from FHM.
Table 1: Summary of results from Single-Laboratory Validation results
Performance characteristics
Aim1
Selectivity
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 levels
Accuracy (Recovery)
Ideally 50-200%
Repeatability Precision RSDr
<20%
Limit of Detection (LOD)
<10 ng/ml
Limit of Quantification (LOQ)
<10 ng/ml
Sample LOQ
200 – 500 ng/ml
(=LOQ x necessary matrix dilution)
Comparison with Biosense
Carp Vtg ELISA kit
Comparison with competitor
FHM Vtg ELISA kit
1)
Goksøyr et al 2003
Value
No response at minimum
dilution = 1:50 (plasma), 1:100
(WBH)
Standard curve working range
0.1-25 ng/ml (250-fold)
75-106%
Within-Day RSDr: 4.5%
Between-Day RSDr: 9.9%
0.02 ng/ml (plasma)
0.04 ng/ml (WBH)
0.09 ng/ml (plasma)
0.11 ng/ml (WBH)
4.68 ng/ml (plasma, 1:50)
11.35 ng/ml (WBH, 1:100)
R2>0.99
R2>0.99
***
INTRODUCTION
Vitellogenin (Vtg) is a large phospholipoglycoprotein, which functions as the egg yolk
precursor in oviparous vertebrates such as fish. The Vtg protein is produced in the
liver and secreted from the liver cells through the secretory pathway, enters the
blood circulation where it is transported to and taken up by growing oocytes.
Endogenous oestrogen levels regulate Vtg production (Figure 1), and plasma Vtg
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Inter-Laboratory validation FHM Vtg ELISA
concentrations normally indicate the maturational status of the female fish (for
reviews, see Mommsen & Walsh, 1988; Arukwe & Goksøyr, 2003). More than a
decade ago, several studies demonstrated that also male fish, caught in rivers and
streams, 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. Since
then, numerous studies have shown the fish Vtg to be a highly responsive
biomarker for estrogenic compounds in both in vitro hepatocyte cell cultures, in vivo
aquaria studies, and field studies (for reviews, see Kime, 1995; Sumpter & Jobling,
1995; Arukwe & Goksøyr, 1998; 2003). Vtg induction in fish has now become an
accepted measure of xenoestrogenic potency of chemicals, effluents, and
discharges.
X
Liver
Vitellogenin
Zona radiata P.
Gonads
Estradiol
Figure 1: In response to oestradiol or xenoestrogens (x), Vitellogenin (egg yolk precursor) and zona
radiata proteins (egg shell precursors) are produced in the liver and transported via the blood to the
gonads.
Within international bodies such as the Organization for Economic Cooperation and
Development (OECD), work is ongoing to develop screening and testing
programmes for endocrine disrupting effects of new chemicals. In the focus of this
development are small fish test species including the fathead minnow (Pimephales
promelas), zebrafish (Danio rerio), and Japanese medaka (Oryzias latipes). These
fish share several attributes that make them ideal test species for reproductive
toxicity 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 methods
for measuring Vtg levels in these fish species, assays that are readily available and
give reproducible results in different laboratories. The enzyme-linked
immunosorbent assay (ELISA) is a sensitive laboratory technique widely used to
detect and quantify antigens in a variety of biological samples. They can be
quantitative (with a standard curve) or qualitative (semi-quantitative - without a
standard curve). The two most widely used principles for quantitative detection of
proteins are the competitive ELISA and the sandwich ELISA techniques (Crowther,
2001).
We have previously developed quantitative sandwich Vtg ELISAs for zebrafish and
Japanese medaka, as well as a homologous carp Vtg ELISA, which also works well
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Inter-Laboratory validation FHM Vtg ELISA
for fathead minnow Vtg (Nilsen et al 2003). Here, we present Single-Laboratory (inhouse) Validation data on a new, homologous quantitative sandwich ELISA for
measuring Vtg in plasma and whole body homogenate samples of the fathead
minnow. The validation has been carried out at Biosense, according to international
guidelines 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 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 FHM
Vtg in the standard or sample added to the ELISA well (Figure 2). After unbound
components are washed away, a Detecting Ab, labelled with the enzyme
horseradish peroxidase (HRP), is added. This Ab binds to a different part of the Vtg
molecule, creating a sandwich of antibodies and Vtg. Addition of the HRP substrate
Tetramethyl Benzidine (TMB) results in a colour reaction where the enzyme
catalyses the conversion of this uncoloured substrate to a blue product. After
development, the reaction is stopped by addition of a mild sulphuric acid, changing
the colour from blue to yellow. The colour intensity is measured using a microplate
reader with a 450 nm filter, and is proportional to the concentration of Vtg in the
standard/sample.
All absorbance levels are corrected for non-specific background reading (NSB), and
a calibration curve is created by plotting Vtg concentration on the x-axis and the
corresponding absorbance level on the y-axis (Figure 3). A 4 parameter or a log-log
curve fit can be used to describe the relationship between the concentration and
the signal. The equation for this calibration curve is then used to calculate the Vtg
concentration in plasma or homogenate samples.
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Inter-Laboratory validation FHM Vtg ELISA
y = 0.1381x1.012
R2 = 0.9988
A450-NSB
10
1
0.1
0.01
0.1
1
10
100
ng/ml Vtg
Figure 3: Fathead minnow Vtg standard curve
***
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 can
also 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.
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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 and
screening, various studies have been conducted over the recent years, involving
both non-commercial laboratory methods and a few commercially available ELISA
kits
(for
zebrafish
Vtg
inter-comparison
study
see
http://abstracts.co.allenpress.com/pweb/setac2003/document/?ID=30012, for an
overview
of
validation
status
in
the
US,
see
http://www.epa.gov/scipoly/oscpendo/assayvalidation/status.htm). These studies
have demonstrated the variance between different Vtg standards, between different
assays and between different laboratories using the same assay.
Clearly, there is a need for standardised, reproducible methods/kits that have been
thoroughly characterised, with validation data describing the assay’s performance,
thus giving the end user reasonable expectancies. The aim of the following study
was to obtain Single-Laboratory Validation data, according to international
guidelines (AOAC, Eurachem, IUPAC).
***
AIMS OF THE VALIDATION STUDY
In a 2003 document, Biosense took the initiative to stress the need for standardised
validations of Vtg standards and assays (Goksøyr et al, 2003). In this document,
based on experience from participation in studies comparing Vtg methods, we
suggested 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 validation
requires the laboratory to select appropriate characteristics for evaluation from the
following: applicability, selectivity, calibration, accuracy, precision, range, limit of
quantification, limit of detection, sensitivity and ruggedness” (Thompson et al 2002).
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Table 2 Pre-determined performance criteria (Goksøyr et al 2003)
Performance characteristics
Aim
Selectivity
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 levels
Accuracy1)
Ideally 50-200%
Repeatability 2)
<20%
Reproducibility3)
<50%
Limit of Detection (LOD)
<10 ng/ml
Limit of Quantification (LOQ)
<10 ng/ml
Sample LOQ
200 – 500 ng/ml
(=LOQ x necessary matrix dilution)
1)
Referred to in this document as Recovery
Referred to in this document Within-Day and Between-Day Repeatability Precision RSDr
3)
Referred to in this document Between-Laboratory Reproducibility Precision RSDR
2)
This study aimed to obtain Single-Laboratory Validation data on the following
parameters:
4. Applicability
5. Calibration
6. Limit of detection (LOD), Limit of Quantification (LOQ)
7. Selectivity
8. Precision (Within-Day, Between-Day)
9. Accuracy (spiking/recovery, bias)
10. Ruggedness
In addition, comparisons with existing methods were also conducted.
***
DEFINITIONS
The terminology involved in validation work is often confusing, and depends largely
on which set of guidelines one looks at. The following definitions have been used in
this report (see http://www.aoac.org/intaffairs/analytical_terminology.htm):
Applicability (Scope): The analytes, matrices and concentrations for which a method
may be used satisfactorily.
Selectivity: The ability to measure accurately the analyte (Vtg) in the presence of
components that may be expected to be present in the matrix (plasma and whole
body homogenate).
Calibration, is the empirical determination of the relationship between the parameter
measured (e.g. ELISA absorbance) and the analyte (Vtg) concentration. The range
of concentrations of analyte where such relationship is established is often referred
to as "calibration range" or the "standard curve working range".
Accuracy is the closeness of agreement between a test result and the accepted
reference value of the property being measured.
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Recovery is the proportion of the amount of analyte, present in or added to, the
analytical portion, which is extracted and presented for measurement
Bias is the difference between the test results and an accepted reference value
Precision is the closeness of agreement between test results obtained under
stipulated conditions.
• Repeatability Precision (same laboratory and operator, samples, equipment,
short time intervals), separated into Within-Day and Between-Day
Repeatability 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 reliably
distinguished from zero. Defined here as reagent blank + 3x standard deviation of
reagent blank. Indicates that the analyte is present, but not necessarily allowing
exact quantification.
Limit of Quantification A concentration above which the analytical method can
operate with an acceptable precision. Defined here as reagent blank + 10x standard
deviation of reagent blank.
Sample LoQ. The LoQ corrected for minimum dilution factor necessary to avoid
matrix effect.
Ruggedness. The ability of the measurement process to resist changes in results
when 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, but
also the Vtg standard used. Here we describe the purification, quality assurance
and 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 in
quantitative assays. In the case of a Vtg ELISA, this CRM should consist of intact
Vtg purified to apparent homogeneity from the test species in question, and should
be quantified according to accepted methods for protein quantification. In addition,
the Vtg should be stabilized for shipping and storage.
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Inter-Laboratory validation FHM Vtg ELISA
Due to the lack of guidelines or criteria for these issues, we will not refer to the FHM
Vtg 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, including
plasma, WBH, liver homogenate, ascites fluid, and egg yolk. For each of these
sample types, the state of the Vtg will be different. For example, the liver cell will
contain immature (unprocessed) Vtg that has not undergone full post-translational
modifications, as well as mature Vtg ready for secretion, whereas the egg yolk will
contain the lipovitellin form processed after uptake. WBH will contain a mixture of
these (unless ovaries and /or liver have been removed), in addition to a high level of
proteolytic activity that may act to degrade the protein during preparation.
We chose to purify Vtg from plasma obtained from estrogenized FHM, because
such 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 to
17ß-oestradiol (or another given reference oestrogen) for a given period of time. A
suitable protease inhibitor (e.g. aprotinin or a protease inhibitor cocktail) should be
added 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 as
possible while maintaining the integrity of the protein and yielding a pure product.
Various methods include
•
•
•
Ion exchange chromatography, with or without a following gel permeation
clean-up (e.g. Brion et al., 2002).
Selective precipitation of Vtg from plasma using MgCl2. This is a rapid
method that has been successfully used with some species (e.g. trout and
carp: Norberg & Haux, 1985, and Nilsen et al., 2003), but appears to be less
useful for other species.
Immunoaffinity-based procedures (e.g. chromatography or magnetic beads).
This strategy requires Vtg-specific Abs, and may give a bias in the
composition of the purified product depending on the epitope specificity of
the antibody used.
Different purification protocols may lead to different compositions of the Vtg holoprotein (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 either
of these methods, mg quantities of purified Vtg can be obtained within a short
period of time (10-30 min). To prevent degradation of the protein this process
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Inter-Laboratory validation FHM Vtg ELISA
should be performed in a cold environment using cold buffers containing protease
inhibitors.
We chose to use anion-exchange chromatography to purify the RM for the FHM Vtg
ELISA. Using spin columns from Vivascience (Hannover, Germany), Vtg was rapidly
purified in sufficient quantities from small amounts of plasma from estrogenized
FHM.
***
QUALITY ASSURANCE OF RM
The purity and homogeneity of the RM needs to be established using gel
electrophoresis. Both SDS-PAGE and 2-dimensional electrophoresis (2-DE) were
carried out to demonstrate the purity of the RM (Figure 4). In addition, Matrix
assisted laser desorption ionization mass spectrophotometry (MALDI-MS) was
performed 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 bands
are clearly visible. All three bands are recognised by the FHM Vtg-specific mAb 1E9
in 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.matrixscience.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 the
main 150 kDa Vtg holoprotein. “FHM3” was not recognised as Vtg, but must be
seen as an unknown, low-molecular weight peptide contaminant.
All in all, the results show that the purified protein is fathead minnow Vtg, and that
the preparation is satisfactory pure.
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Inter-Laboratory validation FHM Vtg ELISA
a
b
1
kDa
2
3
4
5
6
7
250
150
100
75
50
37
c
kDa
FHM1
FHM2
97.4
66.2
FHM3
Figure 4 a: SDS-PAGE with different amounts of FHM Vtg. 7% separation gel. Lane 1: Broad range
standard (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 Vtg
Figure 4 c: 2-DE of FHM Vtg. 50 µg Vtg was applied. pH 3-10, 9% separation gel, low range
standard (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 on
a pure product.
•
Staining methods such as Lowry or Bradford rely on the use of a protein
standard, normally bovine serum albumin (BSA), and assume a similar
staining response of the protein in question to this standard protein. In many
cases, this may not be true; especially for a protein like Vtg, which contains
various non-peptide groups (sugars, phosphates, lipids).
Page 28
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
•
•
Measurement of absorbance at 280 nm is a simple and commonly used
quantification method. The method relies on the use of an extinction
coefficient, which is different for each protein, due to the intramolecular
environment affecting the exposure of UV absorbing aromatic amino acid
residues. A theoretical extinction coefficient can be calculated from the full
amino acid sequence of a protein, however posttranslational modifications
will affect this value.
A more precise method of protein quantification is quantitative amino acid
analysis. This analysis can be performed by independent analytical
laboratories using standard instrumentation. The amino acid composition can
be compared to the theoretical composition if the sequence is known. A
limitation of this method is that only the protein portion of the Vtg molecule is
quantified. The lipid and phosphate parts have been reported for some
species 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 the
limitations, and we chose this method for quantification of the FHM Vtg RM. Two
parallel analyses were performed at the Peptide Synthesis Lab at the Biotechnology
Centre of Oslo, Norway.
***
STABILIZATION OF RM
Vtg is sensitive to freeze-thaw cycles (Figure 5), and needs to be stored at –80°C
and shipped on dry ice. In the Biosense laboratory, we have developed a
lyophilisation procedure that gives good stability to purified Vtg, with more than one
year’s stability of Vtg at 4°C (these stability data are “real time” data, and are so far
based on other species than FHM). After lyophilisation, the RM Vtg was calibrated
against non-lyophilised Vtg in the FHM ELISA.
FHM Vtg freeze/thaw cycles
% of theoretical
amount
100
80
60
40
20
0
0
1
2
3
4
5
Numer of freeze/thaw cycles
Figure 5: Stability of lyophilised FHM Vtg during repeated freeze-thaw cycles. One vial of
reconstituted FHM Vtg was exposed to repeated freezing and thawing. The amount of Vtg in the vial
was analysed in the FHM Vtg ELISA.
Page 29
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
The FHM Vtg RM was compared to FHM Vtg supplied as a standard for the US
EPA method study (http://www.epa.gov/scipoly/oscpendo/assayvalidation/status.htm).
This Vtg had been purified using anion-exchange chromatography and had also
been quantified using amino acid analyses, but the Vtg solution had been thawed
once before this comparison was made. Figure 6 shows that there are between 1.2
and 1.6-fold difference between the absorbance values obtained with the two
standards. This small difference is most likely due to the state of degradation and
purity of the Vtg.
FHM standards
10
y = 0.2018x0.9864
A450-NSB
R2 = 0.9992
y = 0.1514x0.9523
1
R2 = 0.9988
Biosense Standard
0.1
US EPA Standard
Power (Biosense Standard)
Power (US EPA Standard)
0.01
0.01
0.1
1
10
100
ng/ml
Figure 6: Comparison of the FHM Vtg RM with FHM Vtg supplied by the US EPA for Vtg assay
comparison 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 having
non-detectable levels of Vtg at a 1:100 dilution were pooled.
WBH from male fathead minnows was a gift from Robert Bringolf, Iowa State
University, 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 of
purified, non-lyophilised Vtg. Samples were mixed thoroughly, aliquoted into suitable
volumes and frozen at –80oC. Concentrations in spiked samples were 5, 25 and
125 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 State
University, USA.
***
Page 30
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
VALIDATION RESULTS
APPLICABILITY
Applicability (Scope): The analytes, matrices and concentrations for which a
method may be used satisfactorily.
This validation was performed in order to determine the characteristics of the FHM
Vtg ELISA, developed for analyses of Vtg in the fathead minnow (Pimephales
promelas), 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 (0.1-25
ng/ml).
***
CALIBRATION
Calibration, is the empirical determination of the relationship between the
parameter measured (e.g. ELISA absorbance) and the analyte (Vtg)
concentration. The range of concentrations of analyte where such relationship is
established is often referred to as "calibration range" or the "standard curve
working range".
Calibration of the assay was performed with purified, lyophilised FHM Vtg RM. A
standard serial dilution containing 11 concentration points was used, and a log-log
curve fit (using Microsoft Excel) was used to define the relationship between
concentration and response (absorbance).
Standard curves from 15 assays were compiled (Figure 6, Table 3 a-b). Three
standard curves were run each day for five days. The standard serial dilutions were
prepared fresh each day, and the same solution was used on three different ELISA
plates. The Within-Day and Between-Day Precision was calculated (Figure 6).
FHM Vtg standard
100
10
90
80
70
60
50
0.1
40
%CV
A450-NSB
1
30
0.01
20
10
0.001
0.01
0
0.1
1
10
100
ng/ml
Standard
Within-Day Precision
Between-Day Precision
Figure 6: Combined FHM Vtg standard curve, average from 15 assays (using Microsoft Excel). The
secondary y-axis shows the Within-Day and Between-Day Repeatability Precisions (RSDr) for the
standard curves.
Page 31
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Table 3a: Within-Day Repeatability Precision for FHM Vtg standard curves
Within 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
%CV
0.061
0.069
0.072
0.084
0.108
0.167
0.276
0.507
0.918
1.822
3.338
5.307
8.2
11.1
8.7
5.3
4.2
4.4
4.0
4.5
4.4
2.2
2.8
16.1
Average absorbance (450 nm),
3 standard curves
%CV
0.060
0.065
0.072
0.083
0.110
0.166
0.278
0.506
0.940
1.811
3.194
5.793
9.0
5.9
5.3
6.2
8.7
10.2
10.1
11.5
11.0
10.0
8.7
7.3
Average absorbance (450 nm),
3 standard curves
%CV
0.057
0.062
0.069
0.079
0.102
0.153
0.255
0.460
0.849
1.657
3.027
5.263
5.3
3.3
5.7
3.6
3.2
4.6
6.5
6.6
5.7
5.8
5.2
13.9
Average absorbance (450 nm),
3 standard curves
%CV
0.055
0.060
0.066
0.074
0.094
0.140
0.215
0.416
0.735
1.444
2.657
4.358
5.6
5.0
5.9
2.3
3.7
5.8
5.1
6.3
5.2
5.1
4.2
12.4
Average absorbance (450 nm),
3 standard curves
%CV
0.056
0.063
0.069
0.082
0.112
0.170
0.271
0.498
0.945
1.661
3.009
5.017
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
Day 2
Day 3
Day 4
Day 5
Table 3b: Between-Day Repeatability Precision for FHM Vtg standard curves
Between-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 on
different 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-log
transformation of the data.
***
LOD AND LOQ (SENSITIVITY)
Limit of Detection is the smallest amount or concentration that can be reliably
distinguished from zero. Defined here as reagent blank + 3x standard deviation of
reagent blank. Indicates that the analyte is present, but not necessarily allowing exact
quantification.
Limit of Quantification A concentration above which the analytical method can
operate with an acceptable precision. Defined here as reagent blank + 10x
standard deviation of reagent blank.
Page 32
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Sample LoQ. The LoQ corrected for minimum dilution factor necessary to avoid matrix
effect.
Limit of Detection (LoD) and Limit of Quantification (LoQ) were determined from
matrix blanks analysed during Precision studies. Since the samples analysed at a
1:100 dilution did not have detectable levels of Vtg, LoD and LoQ were determined
from NSB-corrected absorbance levels and “translated” into concentration using the
relevant standard curve equation (Table 4a-c).
Table 4a) LoD and LoQ for Plasma
Run1
Replicate 1
A450nm
0.000
Replicate 2
A450nm
-0.003
Replicate 3
A450nm
-0.002
Average
A450nm
-0.001
Stdev
A450nm
0.002
3x Stdev
A450nm
0.003
LoD
ng/ml
0.02
10x Stdev LoQ
A450nm ng/ml
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
0.003
0.02
0.013
0.09
Average
Table 4b) LoD and LoQ for WBH
Run1
Replicate 1
A450nm
-0.004
Replicate 2
A450nm
0.000
Replicate 3
A450nm
-0.001
Average
A450nm
-0.002
Stdev
A450nm
0.002
3x Stdev
A450nm
0.005
LoD
ng/ml
0.04
10x Stdev LoQ
A450nm ng/ml
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
0.002
0.04
0.014
0.11
Average
Table 4c) LoD and LoQ for Dilution buffer
Run1
Replicate 1
A450nm
-0.002
Replicate 2
A450nm
0.001
Replicate 3
A450nm
-0.006
Average
A450nm
-0.002
Stdev
A450nm
0.004
3x Stdev
A450nm
0.009
LoD
ng/ml
0.06
10x Stdev LoQ
A450nm ng/ml
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
0.002
0.03
0.013
0.09
Average
The LoD varied between 0.02 and 0.04 ng/ml for different sample types, and the
LoQ varied between 0.09 and 0.11 ng/ml. Thus, the LoQ is in good agreement with
the 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 on
quantification of Vtg, plasma and WBH were investigated for adverse effects on the
signal response, i.e. plasma or matrix effect. Different dilutions of matrix blanks were
Page 33
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
spiked with a range of Vtg concentrations, and recovery was measured and
compared.
Figure 8 and 9 show that there is an inhibition of the signal at low dilutions of both
plasma and WBH, resulting in an underestimation of Vtg at these dilutions. This
effect varies somewhat between different samples, and with the spike
concentration. Note that some haemolysis had happened during preparation of the
plasma, which may have an effect on matrix effect.
Based on these results showed in Figure 8 and 9, we recommend 1:50 for plasma
and 1:100 for WBH as the minimum dilution factors. Lower dilution factors might be
used, but this should be tested in individual laboratories, and may depend on the
method of sample preparartion.
Plasma
Wbh
b
)
2.5
2.0
1.5
A450-NSB
A450-NSB
a)2.0
1.5
1.0
1.0
0.5
0.5
0.0
0.0
50 100 150 200 250 300 350 400 450 500 550
50
Dilution factor (x-fold)
0
0.5
2.5
100 150 200 250 300 350 400 450 500 550
Dilution factor (x-fold)
0
0.5
2.5
12.5
12.5
Parallelism between standard and
unspiked samples
c)
10
A450-NSB
1
Std
Plasma
0.1
Wbh
Wbh
Power
(Plasma)
Power (Std)
0.01
Power (Wbh)
0.001
10
100
1000
10000
100000
(Relative) dilution factor (x-fold)
Figure 8: Effect of plasma and WBH sample dilution on detection of Vtg. Experiments were
performed with spiked samples (Figure 8a-b) or with naturally incurred samples (Figure 8c),
containing Vtg.
Page 34
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
a)
b
Plasma
Wbh
a)
)
100
ng/ml (measured
ng/ml (measured
100
10
1
0.1
0.01
0.01
0.1
1
10
10
1
0.1
0.01
0.01
100
0.1
PLASMA 1:50
c)
PLASMA 1:100
PLASMA 1:200
d
) 100
Recovery vs dilution
Averagwe recovery (%)
120
100
80
Plasma
Wbh
60
40
20
STANDARD + 5 µl
Average recovery (%)
STANDARD
1
10
100
ng/ml (theoretical)
ng/ml (theoretical)
0
WBH 1:50
WBH 1:100
WBH 1:200
Plasma
80
60
Plasma
40
20
0
50
100
200
25
Dilution factor
50
100
Dilution factor
Figure 9: Effect of plasma and WBH sample dilution on quantification of Vtg. Different amounts of
plasma or WBH were added to standard dilutions (Figure 9a-b). The average recovery for each
sample dilution factor was cacluated (Figure 9c). An additional experiment was conducted with even
lower 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 stipulated
conditions.
• Repeatability Precision (same laboratory and operator, samples, equipment, short
time intervals), separated into Within-Day and Between-Day Repeatability
precision, usually expressed as relative standard deviation, RSDr.
Matrix blanks from plasma and WBH, as well as ELISA kit Dilution buffer were
analysed, spiked with three different concentrations of Vtg corresponding to the
low, 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 and
analysed in triplicates at a 1:100 final dilution.
Within-Day and Between-Day Repeatability Precision (RSDr), often referred to as
intra- 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.
Page 35
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Overall Within-Day Repeatability Precision: average of the individual Precision values for five
days
Table 5a) Within-Day Precision for spiked Plasma samples
Run #
1
2
3
4
5
Spike concentration
ng/ml
0.5
Replicate 1
ng/ml
0.3
Replicate 2
ng/ml
0.3
Replicate 3
ng/ml
0.3
Average
ng/ml
0.3
RSDr
%
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
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
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
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
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 samples
Run #
1
2
3
4
5
Spike concentration
ng/ml
0.5
Replicate 1
ng/ml
0.3
Replicate 2
ng/ml
0.3
Replicate 3
ng/ml
0.3
Average
ng/ml
0.3
RSDr
%
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
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
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
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
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
Overall Within-Day Precision (%)
4.6
Table 5c) Within-Day Precision for spiked Dilution buffer samples
Page 36
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Run #
1
2
3
4
5
Spike concentration
ng/ml
0.5
Replicate 1
ng/ml
0.5
Replicate 2
ng/ml
0.4
Replicate 3
ng/ml
0.4
Average
ng/ml
0.4
RSDr
%
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
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
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
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
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 (%)
4.6
Overall Within-Day Precision for all sample types and concentrations
4.5
Between-Day Repeatability Precision: % CV between the average measured concentrations
from each day.
Overall Between-Day Repeatability Precision: Average Precision for all concentrations
Table 6a) Between-Day Precision for Plasma samples
Spike concentration
ng/ml)
0.5
Average
(ng/ml)
0.3
RSD
%
11.5
2.5
1.8
8.2
12.5
10.3
8.8
r
Overall Between-Day Precision
9.5
Table 6b) Between-Day Precision for WBH samples
Spike concentration
ng/ml)
0.5
Average
(ng/ml)
1.8
RSD
%
1.9
2.5
1.6
10.3
12.5
5.8
17.4
r
Overall Between-Day Precision
9.9
Page 37
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Table 6c) Between-Day Precision for Dilution buffer samples
Spike concentration
ng/ml)
0.5
Average
(ng/ml)
0.4
RSD
%
14.7
2.5
2.1
11.4
12.5
11.1
4.7
r
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 (Overall
Within-Day Precision = 4.5 %) and between successive days (Overall Between-Day
Precision = 9.9 %).
***
ACCURACY
Accuracy is the closeness of agreement between a test result and the accepted
reference value of the property being measured.
Recovery is the proportion of the amount of analyte, present in or added to, the
analytical portion, which is extracted and presented for measurement
Bias is the difference between the test results and an accepted reference value
Recovery and bias
The concentrations measured in the spiked samples during Precision studies were
compared to the theoretical values and Recovery and Bias were determined using
the following formulas (Table 7-8):
Recovery = (C1-C2)/C3 x 100
Where C1= concentration measured in spiked sample, C2= concentration measured in unspiked
sample, C3= theoretical concentration.
Bias = (C3-(C1-C2))/C3 x 100
Where C1= concentration measured in spiked sample, C2= concentration measured in unspiked
sample, 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 (%)
Page 38
74.7
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
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 spike
concentration, with an overall 25% underestimation of spike concentrations in the
sample.
Using freshly spiked samples, without previous freezing/thawing of the samples,
Recovery was measured during the Selectivity study (see Figure 9). Results from
these experiments, using several different concentrations of Vtg (standard curve
dilution series), and different dilutions of plasma and WBH, gave the Recovery
results 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
***
RUGGEDNESS
Ruggedness. The ability of the measurement process to resist changes in results
when subjected to minor changes in environmental and procedural variables.
In order to investigate the stability of the assay when exposed to variations in the
environment and assay procedure, seven parameters were combined in eight
assays to determine their effect on quantification in the FHM Vtg ELISA (according
to Youden & Steiner, 1975).
The effect on quantification of the three matrix blanks, spiked with three different
concentrations, was analysed. Table 10 shows the compiled results.
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Table 10: Ruggedness test. Standard conditions are shown in bold. Alterations higher than 10% are
highlighted.
Condition altered
Value of
condition
Sample type
Sample concentration level
low
med
Difference (%)
Buffer temperature
RT2
30 C
Incubation temperature
o
Standard/sample incubation time
Detection Ab incubation time
4
6
-10
-13
-11
WBH
4
-9
-12
-5
Buffer
12
11
4
9
Plasma
-18
-18
-21
-19
WBH
-7
-5
-8
-7
Buffer
-2
1
7
2
Plasma
6
7
11
8
WBH
1
4
8
4
Buffer
5
4
-3
2
Plasma
-1
-10
-11
-7
WBH
3
1
-5
0
Buffer
8
3
-6
2
Plasma
16
11
-1
9
WBH
11
7
-5
4
Buffer
7
0
1
3
Plasma
9
8
4
7
WBH
4
3
1
3
Buffer
-8
-5
1
-4
Plasma
-2
1
6
2
WBH
-7
-4
0
-3
RT
4C
o
Development time
6
-11
5
3
TMB solution temperature
7
Plasma
0.5 h
1h
before
Buffer
1.5 h
1h
washes
Average (%) 1
Cold
RT
Number of
development
high
1
20 min
30 min
1
Minus sign denotes that the value for the unaltered condition was lower than for the altered
condition
2
RT in this assay was 23oC
The results show that buffer and incubation temperature have the highest influence
on Vtg quantification in the FHM Vtg ELISA. The effect of buffer temperature was
studied in separate assays, and showed that room-tempered buffer had a stronger
effect on the samples than on the standard, increasing absorbance and the
measured Vtg concentration in the samples (data not shown). These results
demonstrate the importance of keeping the Dilution buffer cold and to keep the
room temperature relatively low (20-25oC). On the other hand, deviations to the
incubation times have less influence on the quantification of samples.
***
Page 40
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
COMPARISON WITH EXISTING METHODS
No established reference method for measuring FHM Vtg exists today, so the new
FHM Vtg ELISA was therefore compared with two other commercially available
ELISA 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 been
tested in the Biosense Carp Vtg ELISA, using both the kit (carp) Vtg standard and
the FHM Vtg standard supplied with the study samples. Figure 10a and 10d show
the results compared. The Carp Vtg ELISA utilises one monoclonal and one
polyclonal carp Vtg-specific antibodies, as well as carp Vtg standard, and the assay
is therefore heterologous for the FHM. However, the antibodies show excellent
cross-reactivity with FHM Vtg (Nilsen et al 2004), reflected in values varying less
than 2-fold between the two assays and a good correlation between Vtg levels
measured in the samples (R2 > 0.99).
***
Competitor FHM ELISA kit
The Biosense and a competitor FHM Vtg ELISA kit were compared using spiked
and unspiked samples with different Vtg levels. Figure 10b, c and e show the two
standard curves and the results from the analysed samples. Although the absolute
values varies on average less than 2-fold, due to differences in standards and
antibodies, the results show very good correlation (R2 > 0.99). The largest difference
is the sensitivity of the assays, with a 78 times more sensitive standard curve in the
Biosense FHM ELISA, and a corresponding lack of detection of Vtg in samples
spiked with low Vtg concentrations in the competitor ELISA kit.
***
Page 41
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Plasma samples
a)
1000000
µg/ml in plasma
100000
10000
1000
100
10
1
0.1
A
B
E
G
FHM ELISA
4488
463.9
303.3
6943
C ELISA, C standard
6719
169.0 0.326 522.5
D
6388
R
S
0.255
4418
49773 48807 52006 507.4
25156 0.252 0.209 0.253 0.310 20073 30526 0.259
H
I
K
L
M
N
P
6727
67459 85811 58270 318.8
0.427 0.272 0.222 0.399
T
U
W
Y
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
Sample ID
b)
Standard curves
10
y = 0.1443x1.0271
R2 = 0.9986
A450-NSB
1
Biosense
Competitor
0.1
Power (Biosense)
0.01
Power (Competitor)
y = 0.0039x1.0359
0.001
0.01
0.1
1
10
100
R2 = 0.9986
1000
ng/ml Vtg
Biosense FHM Vtg ELISA vs competitor FHM ELISA
c) 10000000
1000000
100000
ng/ml
10000
1000
Biosense
100
Competitor
10
1
0.1
PJ
Biosense
PQ
PB
PS
0.36
2.12
11.4
WJ
Competitor
WB
1.54
WS
8.66
BJ
BQ
BB
BS
WM
WA
WR
0.35
2.16
11.4
6.89
25.2
45011
996
38334 46661
84219
1956
84441 74427
14.63
100000
d)
WQ
0.30
21.5
36.9
PA
PR
e)
2
R = 0.9937
10000000
10000
2
R = 0.9911
1000000
100000
Wbh samples
Plasma samples
100
Power (Wbh samples)
Power (Plasma samples)
10
Competitor kit
1000
Carp kit
PM
10000
R2 = 0.9988
1000
100
10
1
1
0.1
0.1
0.1
0.1
1
10
100
1000
10000
1
10
100
1000
10000 100000 1E+06 1E+07
Biosense kit
100000
FHM kit
Figure 9: Comparison of the Biosense FHM Vtg kit with two methods. Figure 9a: Samples from a US
EPA 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 the
Biosense and competitor FHM Vtg ELISA kit. Figure 9c: Identical samples analysed in the Biosense
and competitor FHM Vtg ELISA kit. 9d: Correlation between results obtained with Biosense Carp and
FHM Vtg ELISA kits. 9e: Correlation between Biosense and competitor FHM Vtg ELISA kits.
***
Page 42
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
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 from
FHM plasma to be used as standard in the ELISA,. The Vtg was further
characterised and quantified, and was found to be of sufficient purity to be used
reliably as Reference Material (RM) in the FHM ELISA.
A Single-Laboratory Validation was performed to validate the performance of the
new Biosense FHM Vtg ELISA. A set of plasma, WBH and ELISA kit Dilution buffer
samples, both spiked, unspiked and naturally incurred (containing Vtg), were
analysed on different days. Variation within days and between days was
determined, as well as the ability of the assay to measure correct values in spiked
samples.
A calibration curve was run, prepared from a serial dilution of the FHM Vtg RM, in
each assay. Using log-log transformation of the standard curves, the working range
was from 0.1-25 ng/ml (250-fold). For 15 standard curves (three separate standard
curves per day for five days), the Within-Run and Between-Run Repeatability
Precision (RSDr) within the working range was 6.0 and 7.7%, respectively. This
shows that the variability between different vials of Vtg RM is low, as is the variability
between standard curves prepared on different days. Also, the 250-fold working
range is well within our aim (100-fold, Goksøyr et al, 2003) Vtg concentrations in
samples can vary over several orders of magnitude, and a broad working range is
important because fewer dilutions from each sample of unknown Vtg concentration
are necessary in order to “hit” the standard curve working range.
Important parameteres for a quantitative assay is the Limit of Detection (LoD) and
Limit of Quantification (LoQ). The FHM assay is highly sensitive, with an average LoD
of 0.03 ng/ml and an LoQ equal to the lower limit of the standard curve working
range, 0.10 ng/ml. With minimum recommended sample dilutions of 1:50 for
plasma 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 EPA
Study) 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 different
days. Within-Day RSDr values were between 3.8 and 4.6 % for the different sample
types, with an overall RSDr of 4.5%. The Between-Day RSDr was between 9.5 and
10.3%, with an overall RSDr of 9.9%. These values show that the assay shows little
variability 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 by
comparing concentrations measured in the ELISA with the theoretical
concentrations. Samples with three different concentrations of Vtg were analysed,
Page 43
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
and the Recovery varied between 63 and 91%, depending on concentration and
sample type, with an overall Recovery of 75% (Bias = -25%).
Vtg is an unstable molecule, prone to degradation (Arukwe and Goksøyr 2003). For
this reason, spiking of samples should ideally be done on the day of analysis to
avoid freeze-thaw cycles and degradation. However, in order to obtain data for
determination of Between-Day Repeatability Precision, samples had to be diluted,
aliquoted and frozen. This is likely to have affected the Recovery of Vtg in spiked
samples. Spiking/recovery experiments performed without freezing and thawing of
the 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 incubation
time and temperatures affected the FHM ELISA’s ability to quantify Vtg to different
degrees. Seven factors were modified and tested together in different combinations
according to Youden & Steiner (1975), and the factors having highest impact on Vtg
quantification were elevated buffer and room temperatures.
Since no official Reference Method for FHM Vtg quantification is established, the
new Biosense FHM ELISA was compared to two exisiting Vtg ELISA kits. The
Biosense Carp Vtg ELISA, although heterologous to the FHM, has successfully
been applied on samples from FHM (Nilsen et al, 2004). A set of FHM samples from
the 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 the
correlation (R2) between the results were >0.99, showing that the two kits give
comparable results. An alternative FHM ELISA kit was also tested, using a set of
spiked and unspiked samples. The Biosense FHM kit standard curve was 78-fold
more sensitive than the competitor ELISA Vtg standard, but the absolute values
varied less than 2-fold and the correlation between the results from the two kits was
equally good, with R2>0.99.
The results from these two comparisons show that although absolute values may
differ, due to factors such as differences in antibodies, Vtg standard quantification,
purity, the assays still yield similar correlating results and suit their purpose of
differentiating between control and exposed groups of fish in studies of endocrine
disruption.
***
CONCLUSION
A successful Single-Laboratory Validation was performed on the Biosense FHM Vtg
ELISA. 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 in
the fathead minnow.
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Table 11: Summary of Single-Laboratory Validation results for the Biosense FHM Vtg ELISA kit
Performance characteristics
Aim1
Value
Selectivity
Matrix blank < LOD
No response at minimum dilution
(with the necessary dilution factor Minimum dilution =
to avoid matrix effects)
1:50 (plasma), 1:100 (WBH)
Calibration
Standard curve working range Standard curve working range
>10-fold, preferably 50-100 fold 0.1-25 ng/ml (250-fold)
to be practical with the dynamic
range found in Vtg levels
Accuracy (Recovery)
Ideally 50-200%
75%2)
79-106%3)
4)
Repeatability
<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
200 – 500 ng/ml
4.68 ng/ml (plasma, 1:50)
(=LOQ x necessary matrix dilution)
11.35 ng/ml (WBH, 1:100)
1)
Goksøyr et al 2003
2
) From Precision studies, samples frozen and thawed once, dilution 1:100
3
) From Selectivity studies, samples freshly spiked and not frozen, dilutions 1:50-1:100
4)
Referred to in this document Within-Day and Between-Day Repeatability Precision, RSDr
***
REFERENCES AND LINKS
AOAC “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 in
fish. Sarsia 83:225-241.
Arukwe A, Goksøyr A (2003). Eggshell and egg yolk proteins in fish: hepatic proteins for the
next generation. Oogenetic, population, and evolutionary implications of endocrine
disruption. Comp Hepatol 2:4.
Brion F, Nilsen BM, Eidem JK, Goksøyr A, Porcher JM (2002). Development and validation
of an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio
rerio). 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 fish
vitellogenin assays as an endpoint in endocrine disruptor testing and screening – a
proposal. http://www.biosense.com/Docs/GoksoyrEtal2003.pdf
Jobling S, Nolan M, Tyler CR, Brighty G, Sumpter JP (1998). Widespread sexual disruption
in wild fish. Environ Sci Technol 32: 2498–2506.
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
Kime DE (1995). The effects of pollution on reproduction in fish. Rev Fish Biol Fisheries
5:52-96
Mommsen TP, Walsh PJ (1988). Vitellogenesis and oocyte assembly. In: Hoar WS, Randall
VJ (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 species
used in endocrine disruptor screening. Anal Bioanal Chem 378:621
http://www.springerlink.com, DOI 10.1007/s00216-003-2241-2
Norberg B, Haux C (1985). Induction, isolation and a characterization of the lipid content of
plasma 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). Estrogenic
effects of effluents from sewage treatment works. Chem Ecol 8: 275-285.
Silversand C, Haux C (1995). Fatty acid composition of vitellogenin from four teleost
species. J Comp Physiol 164B: 593-599.
Single Laboratory Validation of Analytical Methods for Dietary Supplements (2003). AOAC
International Training Course in Atlanta, Georgia, USA
Sumpter JP, Jobling S (1995). Vitellogenesis as a biomarker for estrogenic contamination of
the 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 methods
of 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.
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
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 following
immediately upon receipt - please note:
1. The state of the samples – samples were shipped on dry ice. There should be dry ice left
in 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 janne@biosense.com
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 plates
each. There are also two vials of Dilution buffer concentrate and TMB substrate, and two vials
of 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 both
assay days.
A Study Protocol is sent as a s eparate file. This contains an o verview of enclosed samples, a
study protocol with plate layouts and Report Sheets. Please return Report and Result sheets
electronically to janne@biosense.com.
Again thank you for your interest to participate in the validation, and good luck with the
analysis!
All the best
Janne K. Eidem
Janne K. Eidem
Scientist, R&D
Biosense Laboratories AS
Thormøhlensgate 55
NO-5008 Bergen, Norway
Tel: +47 55 54 39 80/66
Mob: +47 91 81 77 81
Fax: +47 55 54 37 71
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
C: STUDY PROTOCOL AND REPORT SHEET
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
***
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Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
D: REPORT SHEET (EXCEL)
Result sheet day 1
Instructions
1. Paste values into each plate (Edit - Paste special - Values)
2. Remove clear outliers (if %CV > 10)
3. Adjust standard curves
4. Use equation from each standard curve to calculate Vtg concentrations in samples
Day 1
A
B
C
D
E
F
G
H
Plate 2
1
0.055
0.055
0.055
0.055
0.103
0.324
1.588
0.112
2
0.060
0.060
0.060
0.054
0.104
0.322
1.613
0.128
3
0.066
0.064
0.065
0.057
0.101
0.332
1.575
0.097
4
0.081
0.080
0.078
0.053
0.102
0.316
1.551
0.130
5
0.104
0.107
0.107
0.052
0.099
0.302
1.543
0.043
6
0.176
0.170
0.163
0.052
0.105
0.315
1.562
0.122
7
0.294
0.281
0.273
0.053
0.106
0.301
1.609
0.042
8
0.542
0.529
0.494
0.053
0.101
0.318
1.596
0.045
9
0.994
0.941
0.933
0.053
0.100
0.321
1.565
0.039
10
1.831
1.833
1.856
0.054
0.053
0.057
0.053
0.126
11
3.445
3.283
3.294
0.054
0.056
0.053
0.054
0.044
12
out 6.000
4.219
4.134
0.054
0.055
0.059
0.058
0.044
A
B
C
D
E
F
G
H
Plate 2
1
0.059
0.064
0.064
1.160
0.285
0.100
0.058
0.117
2
0.073
0.069
0.067
1.158
0.274
0.104
0.055
0.098
3
0.073
0.080
0.069
1.141
0.270
0.106
0.062
0.102
4
0.090
0.087
0.079
1.056
0.265
0.107
0.062
0.097
5
0.112
0.115
0.102
1.052
0.263
0.099
0.063
0.098
6
0.171
0.169
0.154
1.013
0.254
0.097
0.063
0.098
7
0.286
0.268
0.261
1.067
0.260
0.097
0.059
0.046
8
0.529
0.496
0.477
1.089
0.263
0.101
0.061
0.043
9
0.943
0.920
0.891
1.051
0.265
0.101
0.063
0.045
10
1.865
1.851
1.758
0.056
0.054
0.060
0.063
0.046
11
3.446
3.320
3.267
0.055
0.058
0.058
0.062
0.044
12
6.000
6.000
6.000
0.056
0.054
0.054
0.058
0.044
NSB
0.055
0.0
0.05
0.060
0.0
0.005
0.10
0.065
1.5
0.010
0.20
0.080
1.9
0.025
0.39
0.106
1.6
0.051
0.78
0.170
3.8
0.115
1.56
0.283
3.7
0.228
3.13
0.522
4.8
0.467
6.25
0.956
3.5
0.901
12.50
1.840
0.8
1.785
25.00
3.341
2.7
3.286
50.00
4.177
1.4
4.122
NSB
0.06233
4.6
0.05
0.07
4.4
0.007
0.10
0.074
7.5
0.012
0.20
0.08533
6.7
0.023
0.39
0.10967
6.2
0.047
0.78
0.16467
5.6
0.102
1.56
0.27167
4.7
0.209
3.13
0.50067
5.3
0.438
6.25
0.918
2.8
0.856
12.50
1.82467
3.2
1.762
25.00
3.34433
2.7
3.282
50.00
6
0.0
5.938
STANDARD CURVE PLATE 1
Concentration (ng/ml)
Absorbance 450 nm
%CV (A450nm)
NSB-corrected absorbance
STANDARD CURVE PLATE 2
Concentration (ng/ml)
Absorbance 450 nm
%CV (A450nm)
NSB-corrected absorbance
y = 0.1324x1.038
Adjusted standard curves
Standard curves
10
10
1
1
R2 = 0.9977
A450-NSB
A450-NSB
y = 0.1283x1.0296
STANDARD CURVE PLATE 1
0.1
STANDARD CURVE PLATE 2
R2 = 0.9996
STANDARD CURVE PLATE 1
0.1
STANDARD CURVE PLATE 2
Power (STANDARD CURVE PLATE 1)
0.01
0.01
Power (STANDARD CURVE PLATE 2)
0.001
0.01
0.001
0.1
1
10
0.01
100
0.1
1
10
100
ng/ml Vtg
ng/ml Vtg
SAMPLES PLATE 1
Sample ID
Dilution factor
Absorbance 450 nm
%CV (A450nm)
NSB-corrected absorbance
Calculated concentration
Corrected for dilution factor
PJ
10
0.055
2.8
0.000
0.002
0.02
PQ
10
0.052
1.1
-0.003
#NUM!
#NUM!
PB
10
0.053
0.0
-0.002
#NUM!
#NUM!
PS
10
0.054
0.0
-0.001
#NUM!
#NUM!
WJ
10
0.103
1.5
0.048
0.346
3.46
WQ
10
0.102
2.9
0.047
0.341
3.41
WB
10
0.102
3.1
0.047
0.344
3.44
WS
10
0.055
2.8
0.000
#NUM!
#NUM!
PM
50
0.326
1.6
0.271
2.103
105.17
SAMPLES PLATE 2
Sample ID
Dilution factor
Absorbance 450 nm
%CV (A450nm)
NSB-corrected absorbance
Calculated concentration
Corrected for dilution factor
BJ
10
1.153
0.9
1.091
9.06
90.57
BQ
10
1.040
2.3
0.978
8.10
80.95
BB
10
1.069
1.8
1.007
8.34
83.40
BS
10
0.056
1.0
-0.007
#NUM!
#NUM!
WM
100
0.276
2.8
0.214
1.69
169.34
WM
10000
0.103
3.0
0.041
0.31
3089.53
WM
1000000
0.058
6.0
-0.004
#NUM!
#NUM!
WA
100
0.261
2.2
0.198
1.57
156.59
WA
10000
0.101
5.2
0.039
0.29
2908.65
PM
PM
5000
500000
1.592
0.112
1.2
13.8
1.537
0.057
12.742
0.419
63711.49 ######
WA
1000000
0.063
0.9
0.000
0.00
2178.34
WR
100
0.263
1.0
0.200
1.58
158.22
PA
50
0.311
2.5
0.256
1.983
99.13
PA
5000
1.552
0.6
1.497
12.398
61991
WR
10000
0.100
2.3
0.037
0.28
2805.44
WR
1000000
0.061
3.3
-0.001
#NUM!
#NUM!
PA
500000
0.098
48.9
0.043
0.314
######
PR
50
0.313
3.4
0.258
2.001
100.07
***
Page 56
PR
5000
1.590
1.4
1.535
12.725
63625.44
PR
500000
0.042
7.1
-0.013
#NUM!
#NUM!
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
E: INDIVIDUAL RAW DATA
LAB 1
Day 1
A
B
C
D
E
F
G
H
Plate 1
1
0.052
0.052
0.054
0.052
0.051
0.059
0.051
0.053
2
0.056
0.057
0.056
0.051
0.052
0.066
0.054
0.054
3
0.063
0.068
0.063
0.051
0.051
0.065
0.056
0.052
4
0.081
0.079
0.078
0.108
0.098
0.095
0.053
0.050
5
0.111
0.107
0.107
0.104
0.096
0.088
0.051
0.051
6
0.176
0.170
0.167
0.105
0.096
0.088
0.051
0.048
7
0.300
0.284
0.287
0.358
0.273
6.000
4.106
0.120
8
0.579
0.553
0.543
0.349
0.276
4.337
3.905
0.119
9
1.097
1.033
0.993
0.349
0.270
4.613
4.023
0.118
10
2.065
2.016
1.930
1.596
1.195
0.052
0.056
0.054
11
3.593
3.429
3.455
1.568
1.230
0.055
0.055
0.052
12
6.000
6.000
4.486
1.618
1.274
0.056
0.052
0.053
A
B
C
D
E
F
G
H
Plate 2
1
0.052
0.053
0.053
0.059
2.637
0.081
0.050
0.050
2
0.058
0.059
0.056
0.049
2.565
0.081
0.050
0.043
3
0.063
0.065
0.060
0.049
2.503
0.078
0.050
0.042
4
0.078
0.081
0.079
0.103
4.070
1.198
0.063
0.044
5
out 0.125
0.107
0.102
0.102
4.783
1.135
0.062
0.042
6
0.174
0.166
0.163
0.102
4.519
1.152
0.062
0.041
7
0.297
0.276
0.263
0.339
4.560
4.606
1.246
0.043
8
0.556
0.515
0.492
0.334
5.560
4.519
1.195
0.042
9
1.034
0.985
0.953
0.338
5.083
4.481
1.195
0.042
10
2.002
1.911
1.820
1.562
0.054
0.052
0.052
0.044
11
3.555
3.452
3.389
1.620
0.055
0.054
0.053
0.041
12
6.000
4.860
4.258
1.655
0.057
0.053
0.053
0.041
A
B
C
D
E
F
G
H
Plate 1
1
0.056
0.055
0.052
0.054
0.062
3.096
0.098
0.057
2
0.061
0.058
0.058
0.052
0.055
3.044
0.091
0.057
3
0.067
0.068
0.063
0.052
0.053
2.955
0.088
0.059
4
0.083
0.078
0.078
0.107
0.094
6.000
1.342
0.073
5
0.114
0.109
0.105
0.107
0.088
4.801
1.339
0.074
6
0.175
0.168
0.163
0.109
0.087
4.217
1.322
0.069
7
0.309
0.290
0.295
0.334
0.252
6.000
4.624
1.538
8
0.568
0.559
0.531
0.342
0.252
4.431
6.000
1.538
9
1.043
1.036
1.009
0.344
0.258
4.430
4.462
1.523
10
1.955
1.968
1.894
1.593
1.195
1.552
0.350
0.113
11
3.455
3.514
3.390
1.627
1.162
1.572
0.345
0.115
12
4.574
4.398
4.796
1.714
1.232
1.546
0.363
0.113
A
B
C
D
E
F
G
H
Plate 2
1
0.053
0.054
0.059
0.051
0.056
0.052
0.051
0.052
2
0.061
0.055
0.061
0.050
0.054
0.050
0.049
0.052
3
0.066
0.064
0.068
out 0.061
0.054
0.052
0.051
0.054
4
0.087
0.079
0.077
0.100
0.087
0.052
0.050
0.050
5
0.115
0.108
0.109
0.114
0.083
0.054
0.050
0.054
6
0.170
0.163
0.158
0.098
0.083
0.054
0.050
0.050
7
0.298
0.279
0.277
0.313
5.264
4.264
0.125
0.054
8
0.519
0.501
0.498
0.313
4.565
4.335
0.123
0.055
9
0.967
0.943
0.927
0.317
4.611
4.286
0.123
0.056
10
1.823
1.829
1.710
1.578
0.380
0.121
0.064
0.057
11
3.635
3.368
3.274
1.642
0.388
0.123
0.072
0.055
12
6.000
6.000
6.000
1.603
0.396
0.125
0.068
0.055
Day 2
Standard curves Day 1 and 2:
y = 0.1443x1.0271
Adjusted standard curves
y = 0.1413x1.0303
Adjusted standard curves
R2 = 0.9986
10
R2 = 0.9984
10
y = 0.1354x1.0401
STANDARD CURVE PLATE 1
0.1
STANDARD CURVE PLATE 2
Power (STANDARD CURVE PLATE 1)
0.01
R2 = 0.9985
1
A450-NSB
A450-NSB
y = 0.1344x1.0253
R2 = 0.9978
1
STANDARD CURVE PLATE 1
0.1
STANDARD CURVE PLATE 2
Power (STANDARD CURVE PLATE 1)
0.01
Power (STANDARD CURVE PLATE 2)
Power (STANDARD CURVE PLATE 2)
0.001
0.01
0.1
1
10
0.001
0.01
100
0.1
1
10
100
ng/ml Vtg
ng/ml Vtg
***
Page 57
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
LAB 2
Day 1
A
B
C
D
E
F
G
H
A
B
C
D
E
F
G
H
Day 2
A
B
C
D
E
F
G
H
A
B
C
D
E
F
G
H
Plate 1
1
0.090
0.092
0.093
0.093
0.095
4.648
0.136
0.105
2
0.116
0.145
0.112
0.104
0.093
4.378
0.140
0.108
3
0.127
0.117
0.112
0.089
0.092
4.334
0.134
0.089
4
0.150
0.133
0.136
0.165
0.138
7.531
1.507
0.110
5
0.201
0.177
0.181
0.173
0.152
7.521
1.561
0.116
6
0.272
0.254
0.236
0.150
0.139
7.541
1.548
0.110
7
0.424
0.399
0.385
0.378
0.354
7.601
7.433
1.608
8
0.686
0.647
0.647
0.380
0.341
7.531
7.452
1.566
9
1.290
1.235
1.176
0.373
0.344
7.452
7.511
1.506
10
2.369
2.279
2.206
1.607
1.395
11
4.617
4.473
4.317
1.602
1.443
12
7.531
7.581
7.601
1.615
1.454
Plate 2
1
0.109
0.090
0.126
0.090
0.097
0.306
0.094
2
0.105
0.103
0.100
0.088
0.112
0.317
0.098
3
0.111
0.114
0.111
0.089
0.103
1.252
0.103
4
0.143
0.138
0.145
0.161
0.201
0.092
0.096
5
0.189
0.181
0.182
0.160
0.148
0.091
0.098
6
0.296
0.276
0.264
0.158
0.169
0.091
0.091
7
0.473
0.455
0.430
0.475
7.683
7.395
0.204
8
0.821
0.785
0.743
0.480
7.672
7.462
0.203
9
1.550
1.489
1.430
0.494
7.632
7.414
0.347
10
2.850
2.733
2.652
2.350
11
5.743
5.396
5.614
2.385
12
7.511
7.601
7.591
2.456
Plate 1
1
0.080
0.303
0.075
0.104
0.101
3.910
0.185
0.117
2
0.316
0.195
0.090
0.131
0.223
3.761
0.263
0.176
3
0.130
0.111
0.119
0.093
0.095
3.935
0.129
0.251
4
0.139
0.997
0.811
0.161
0.162
8.583
1.773
0.117
5
0.193
0.180
0.177
0.163
0.148
7.109
1.738
0.112
6
0.276
0.259
0.249
0.152
0.149
6.962
1.700
0.115
7
0.454
0.446
0.442
0.454
0.386
8.481
7.309
2.050
8
0.874
0.802
0.776
0.469
0.373
7.611
6.639
1.926
9
1.597
1.427
1.420
0.472
0.381
9.175
6.495
1.973
10
3.043
2.707
2.693
2.259
1.836
11
5.478
5.111
4.935
2.255
1.898
12
7.906
7.652
7.704
2.422
1.745
Plate 2
1
0.093
0.128
0.091
0.091
0.089
0.105
0.092
2
0.105
0.110
0.125
0.096
0.090
0.101
0.106
3
0.142
0.116
0.112
0.101
0.093
0.093
0.110
4
0.153
0.135
0.145
0.181
0.125
0.092
0.093
5
0.190
0.180
0.176
0.169
0.124
0.102
0.098
6
0.285
0.283
0.221
0.169
0.125
0.095
0.095
7
0.445
0.421
0.400
0.482
7.873
7.501
0.258
8
0.797
0.728
0.699
0.497
7.190
7.423
0.255
9
1.401
1.299
1.247
0.472
7.581
7.414
0.305
10
2.819
2.571
2.357
2.258
11
4.716
4.453
4.424
2.253
12
7.511
7.611
7.601
2.265
Standard curves Day 1 and 2:
y = 0.2149x0.915
Adjusted standard curves
y = 0.2551x0.9271
Adjusted standard curves
R2 = 0.9992
10
R2 = 0.9988
10
1.0206
y = 0.2094x
y = 0.2218x0.9439
R2 = 0.9993
STANDARD CURVE PLATE 1
0.1
STANDARD CURVE PLATE 2
A450-NSB
A450-NSB
R2 = 0.9986
1
1
Power (STANDARD CURVE PLATE 1)
0.01
STANDARD CURVE PLATE 1
0.1
STANDARD CURVE PLATE 2
Power (STANDARD CURVE PLATE 1)
0.01
Power (STANDARD CURVE PLATE 2)
Power (STANDARD CURVE PLATE 2)
0.001
0.01
0.1
1
10
0.001
0.01
100
0.1
1
10
100
ng/ml Vtg
ng/ml Vtg
***
Page 58
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
LAB 3
Day 1
A
B
C
D
E
F
G
H
Plate 1
1
0.060
0.061
0.061
0.059
0.059
1.591
0.076
0.061
2
0.060
0.060
0.063
0.059
0.059
1.461
0.133
0.061
3
0.063
0.064
0.065
0.060
0.061
1.428
0.077
0.062
4
0.067
0.069
0.071
0.080
0.075
4.000
0.584
0.067
5
0.077
0.077
0.080
0.077
0.073
4.000
0.590
0.066
6
0.098
0.097
0.094
0.076
0.070
4.000
0.577
0.069
7
0.137
0.131
0.132
0.157
0.107
4.000
4.000
0.538
8
0.217
0.209
0.204
0.158
0.145
4.000
4.000
0.535
9
0.370
0.347
0.343
0.161
0.105
4.000
4.000
0.555
10
0.667
0.636
0.631
0.632
0.335
0.061
0.061
0.062
11
1.253
1.180
1.177
0.632
0.342
0.062
0.062
0.060
12
2.271
2.179
2.197
0.694
0.346
0.061
0.062
0.062
A
B
C
D
E
F
G
H
Plate 2
1
0.064
0.059
0.066
0.064
0.064
0.065
0.078
0.069
2
0.066
0.063
0.067
0.066
0.062
0.065
0.078
0.072
3
0.068
0.064
4
0.076
0.070
0.075
0.089
0.066
0.069
0.080
0.073
5
0.084
0.083
0.086
0.087
0.072
0.067
0.084
0.070
6
0.107
0.104
0.109
0.088
0.069
0.069
0.084
0.072
7
0.151
0.150
0.150
0.179
4.000
2.655
0.097
0.067
8
0.248
0.233
0.259
0.180
4.000
2.536
0.097
0.075
9
0.426
0.418
0.415
0.185
4.000
2.532
0.099
0.070
10
0.773
0.732
0.691
0.717
0.065
0.066
0.067
0.067
11
1.321
1.345
1.388
0.715
0.064
0.071
0.072
0.071
12
2.396
2.277
2.435
0.666
0.074
0.069
0.072
0.074
Day 2
0.063
0.064
0.068
0.084
0.069
A
B
C
D
E
F
G
H
Plate 1
1
0.058
0.053
0.056
0.060
0.054
1.350
0.072
0.067
2
0.056
0.053
0.056
0.062
0.053
1.191
0.071
0.058
3
0.057
0.057
0.057
0.056
0.059
1.222
0.068
0.056
4
0.062
0.061
0.062
0.074
0.065
4.000
0.605
0.064
5
0.075
0.072
0.073
0.075
0.074
4.000
0.616
0.071
6
0.097
0.093
0.094
0.078
0.065
4.000
0.639
0.067
7
0.140
0.135
0.138
0.161
0.113
4.000
4.000
0.574
8
0.230
0.209
0.211
0.160
0.122
4.000
4.000
0.566
9
0.386
0.345
0.368
0.160
0.109
4.000
4.000
0.562
10
0.759
0.710
0.699
0.654
0.374
0.058
0.054
0.058
11
1.380
1.259
1.253
0.667
0.377
0.056
0.054
0.057
12
2.501
2.383
2.391
0.718
0.425
0.057
0.056
0.056
Plate 2
1
0.069
0.058
0.062
0.060
0.061
0.071
0.073
0.087
2
A
B
C
D
E
F
G
H
0.059
0.064
0.061
0.073
0.075
0.077
0.083
3
0.072
0.067
0.067
0.061
0.068
0.075
0.083
0.098
4
0.079
0.074
0.076
0.089
0.068
0.069
0.067
0.083
5
0.094
0.083
0.087
0.090
0.066
0.074
0.071
0.087
6
0.133
0.116
0.124
0.092
0.070
0.073
0.067
0.079
7
0.181
0.189
0.176
0.211
4.000
2.321
0.098
0.087
8
0.294
0.258
0.290
0.191
4.000
2.783
0.109
0.088
9
0.478
0.494
0.482
0.188
4.000
2.615
0.103
0.085
10
0.999
0.950
0.937
0.771
0.069
0.076
0.082
0.082
11
1.734
1.598
1.862
0.864
0.061
0.066
0.074
0.075
2.640
2.797
0.885
0.061
0.070
0.079
0.084
12
Standard curves Day 1 and 2:
Adjusted standard curves
y = 0.0491x1.0076
Adjusted standard curves
y = 0.0461x0.9962
R2 = 0.9989
R2 = 0.9997
10
10
y = 0.0704x0.9712
y = 0.0553x0.9793
R2 = 0.999
STANDARD CURVE PLATE 1
0.1
STANDARD CURVE PLATE 2
Power (STANDARD CURVE PLATE 1)
0.01
R2 = 0.9971
1
A450-NSB
A450-NSB
1
STANDARD CURVE PLATE 1
0.1
STANDARD CURVE PLATE 2
Power (STANDARD CURVE PLATE 1)
0.01
Power (STANDARD CURVE PLATE 2)
Power (STANDARD CURVE PLATE 2)
0.001
0.01
0.1
1
10
100
0.001
0.01
0.1
1
10
100
ng/ml Vtg
ng/ml Vtg
***
Page 59
Biosense Laboratories AS
Inter-Laboratory validation FHM Vtg ELISA
LAB 4
Day 1
A
B
C
D
E
F
G
H
A
B
C
D
E
F
G
H
Day 2
A
B
C
D
E
F
G
H
A
B
C
D
E
F
G
H
Plate 1
1
0.068
0.049
0.046
0.044
0.087
0.477
0.060
0.164
2
0.548
0.046
0.048
0.057
0.082
0.401
0.051
0.055
3
0.055
0.049
0.345
0.046
0.086
0.649
0.068
0.105
4
0.223
0.052
0.052
0.064
0.060
3.550
0.454
0.108
5
0.059
0.061
0.060
0.067
0.057
3.439
0.447
0.078
6
0.074
0.196
0.088
0.055
0.090
3.551
0.472
0.070
7
0.107
0.147
0.122
0.083
0.112
3.691
4.000
0.374
8
0.166
0.280
0.180
0.126
0.112
3.731
4.000
0.310
9
0.366
0.352
0.361
0.107
0.204
3.659
4.000
0.383
10
0.546
0.516
0.483
0.407
0.357
11
1.173
0.934
1.070
0.370
0.223
12
1.493
2.003
1.745
0.389
0.327
0.056
0.053
0.073
Plate 2
1
0.146
0.137
0.302
0.155
1.143
0.139
0.263
2
0.075
0.150
0.150
0.088
1.807
2.836
2.283
3
0.104
0.183
0.119
0.106
0.266
0.264
2.297
4
0.102
0.132
0.090
0.146
0.203
1.453
0.101
5
0.135
0.165
0.171
0.092
0.269
0.216
1.420
6
0.155
0.361
0.192
0.071
0.225
0.231
0.186
7
0.161
0.384
0.399
0.214
3.543
1.538
1.133
8
0.209
0.349
0.363
0.176
3.702
1.793
0.113
9
0.457
0.404
0.444
0.140
3.576
1.132
0.078
10
0.594
0.674
0.696
0.440
11
1.212
1.082
1.128
0.375
12
1.900
1.736
2.026
0.394
0.203
0.116
0.065
Plate 1
1
0.447
0.381
0.382
0.168
0.206
3.660
0.291
0.214
2
0.393
0.330
0.327
0.152
0.203
3.678
0.244
2.283
3
0.200
0.214
0.220
0.167
0.366
3.683
0.223
0.279
4
0.309
0.313
0.183
0.245
0.240
3.952
2.245
3.900
5
0.240
0.288
0.265
0.364
0.268
3.944
2.247
0.250
6
0.472
0.524
0.604
0.307
1.087
3.968
2.194
0.245
7
0.569
0.705
0.637
0.746
0.414
4.000
4.000
2.805
8
1.628
0.940
1.373
0.614
0.483
4.000
4.000
2.745
9
1.935
1.620
1.622
0.596
1.825
4.000
4.000
2.679
10
2.677
2.626
2.510
2.570
1.434
11
3.800
3.740
3.709
2.380
1.514
12
4.000
4.000
4.000
2.216
1.394
0.157
0.198
0.368
Plate 2
1
2.064
0.235
0.927
0.087
0.095
0.074
0.155
2
0.287
0.080
0.122
0.190
0.133
0.097
0.071
3
0.139
0.913
3.789
0.072
0.137
0.069
0.065
4
0.173
0.224
1.950
0.160
0.105
0.065
0.139
5
0.242
0.230
0.215
0.158
0.090
0.092
0.104
6
0.361
0.536
0.791
0.157
0.088
0.074
0.059
7
0.440
0.424
0.408
3.699
4.000
4.000
0.230
8
0.763
0.721
0.674
2.045
4.000
4.000
0.224
9
1.108
1.221
1.189
0.451
4.000
4.000
1.087
10
2.101
2.238
2.213
1.973
11
3.582
3.506
3.445
1.790
12
4.000
4.000
4.000
1.790
0.091
0.231
0.144
Standard curves Day 1 and 2:
Adjusted standard curves
2
R = 0.9936
10
y = 0.2757x0.8992
Adjusted standard curves
y = 0.0376x1.0542
R2 = 0.9814
10
0.8386
y = 0.2315x
y = 0.0646x0.8492
R2 = 0.9935
0.1
0.01
R2 = 0.9978
1
A450-NSB
A450-NSB
1
STANDARD CURVE PLATE 1
0.1
STANDARD CURVE PLATE 2
Power (STANDARD CURVE PLATE 1)
0.01
STANDARD CURVE PLATE 1
Power (STANDARD CURVE PLATE 2)
STANDARD CURVE PLATE 2
0.001
0.01
0.1
1
ng/ml Vtg
10
Power (STANDARD
CURVE PLATE 1)
100
0.001
0.01
Power (STANDARD CURVE PLATE 2)
0.1
1
10
100
ng/ml Vtg
***
Page 60