Bead-based ELISA for validation of ovarian cancer early detection markers.

Abstract

Efforts to validate ovarian cancer early detection biomarkers with immunoassays are challenged by the limited specimen volumes available. We sought to develop a specimen-efficient assay to measure CA125 in serum, assess its reproducibility, validity, and performance, and test its potential for multiplexing and combining with human epididymis protein 4 (HE4), a promising novel ovarian cancer marker.
Four pairs of commercially available anti-CA125 antibodies and one pair of anti-HE4 antibodies were evaluated for accuracy in measuring known concentrations of antigen on a bead-based platform. The two best pairs were further assessed for reproducibility, validity, and the ability to discriminate between blinded serum samples obtained from ovarian cancer cases (n = 66) and women without ovarian cancer (n = 125).
Suitability for use in a bead-based assay varied across CA125 antibody pairs. Two CA125 bead-based assays were highly reproducible (overall correlations between replicates >/= 0.95; coefficients of variation < 0.2) and strongly correlated with the research standard CA125II RIA (correlations >/= 0.9). Their ability to distinguish ovarian cancer cases from non-cases based on receiver operating characteristic analyses (area under the curve, AUC, of 0.85 and 0.84) was close to that of the CA125II RIA (AUC, 0.87). The HE4 bead-based assay showed lower reproducibility but yielded an AUC of 0.89 in receiver operating characteristics analysis. Multiplexing was not possible but a composite marker including CA125 and HE4 achieved an AUC of 0.91.
Optimization procedures yielded two bead-based assays for CA125 that perform comparably to the standard CA125II RIA, which could be combined with an HE4 bead-based assay to improve diagnostic performance, and requires only 15 muL of sample each.

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Bead-Based ELISAfor Validation of Ovarian Cancer Early
Detection Markers
Nathalie Scholler,1Meghan Crawford,1Alicia Sato,2Charles W. Drescher,3Kathy C. O’Briant,3
Nancy Kiviat,4Garnet L. Anderson,2and Nicole Urban3
Abstract
Purpose: Efforts to validate ovarian cancer early detection biomarkers with immunoassays are
challengedby thelimited specimenvolumes available.We sought to develop a specimen-efficient
assay tomeasureCA125inserum,assessitsreproducibility,validity,andperformance,andtestits
potential for multiplexing and combining with human epididymis protein 4 (HE4), a promising
novelovariancancermarker.
Experimental Design: Four pairs of commerciallyavailable anti-CA125 antibodies andone pair
ofanti-HE4antibodieswereevaluatedforaccuracyinmeasuringknownconcentrationsofantigen
on a bead-based platform.The two best pairs were further assessed for reproducibility, validity,
andtheabilitytodiscriminatebetweenblindedserumsamplesobtainedfromovariancancercases
(n = 66) and womenwithoutovariancancer (n = 125).
Results: Suitability for use in a bead-based assay varied across CA125 antibody pairs.Two
CA125 bead-based assays were highly reproducible (overall correlations between replicates z
0.95; coefficients of variation < 0.2) and strongly correlated with the research standard CA125II
RIA(correlationsz0.9).Theirability todistinguishovariancancercasesfromnon-casesbasedon
receiveroperatingcharacteristicanalyses(areaunderthecurve,AUC,of0.85and0.84)wasclose
to that of the CA125IIRIA (AUC, 0.87).The HE4 bead-basedassay showedlower reproducibility
but yielded an AUC of 0.89 in receiver operating characteristics analysis. Multiplexing was not
possiblebut a compositemarkerincluding CA125 and HE4 achievedan AUC of 0.91.
Conclusion: Optimization procedures yielded two bead-based assays for CA125 that perform
comparably to the standard CA125IIRIA, which could be combined with an HE4 bead-based
assay toimprove diagnosticperformance, and requires only15 AL of sample each.
Many candidate markers are being evaluated for their use in an
early detection biomarker panel for ovarian cancer (1), but they
have not yet been evaluated in preclinical samples obtained one
or more years prior to diagnosis because such specimens are
very precious. CA125, a high–molecular weight glycoprotein
recognized by antibodies belonging to only three epitope
groups (2–4), is elevated in most women with ovarian cancer
(5). It has been extensively studied (6–10) and is likely to be
included in serum marker panels that are proposed for
validation in preclinical samples. Quantification of serum
CA125 levels is currently based on heterologous assays using
two monoclonal antibodies (mAb) directed against the epitope
groups, M11 and OC125, in contrast with the original
homologous assay using only one mAb directed against the
OC125-like epitope (11). The inadequate sensitivity of CA125
for early stage disease and its poor specificity to malignancy
limit its use for population screening (12–15). Adding one or
several markers to CA125 for use as a composite marker (CM)
would improve performance in a screening program if
sensitivity were improved with no loss in specificity (16–18)
and stability over time yielded better performance in a
longitudinal algorithm (16).
The lead time of a marker is critical, as it measures the
marker’s ability to identify disease early in the disease process.
Repositories developed by the Carotene and Retinol Efficacy
Trial (19), the Women’s Health Initiative (20), and the Prostate,
Lung, Colon, and Ovary Cancer Screening Trial (21) have
preclinical samples for a relatively large number of cases for
whom blood samples were collected well in advance of
diagnosis, making it possible to estimate the lead time of
candidate serum biomarkers. Because of the value and scarcity
of these resources, however, use of these specimens must be
well-justified and optimized. The research standard, CA125 RIA
CA125II, from Fujirebio Diagnostics, Inc. (FDI, Malvern, PA)
requires 0.2 mL of serum sample, limiting the number of other
candidates that can be evaluated simultaneously in a typical
research sample of 0.5 mL. Some clinical assays require less
specimen, but they yield results that vary by type, manufacturer,
Imaging, Diagnosis, Prognosis
Authors’Affiliations:1Translational Outcomes Research Laboratory,2Women’s
Health Initiative Clinical Coordinating Center,3Cancer Prevention Program at the
Fred Hutchinson Cancer Research Center, Public Health Sciences, and
4Harborview Medical Center, University ofWashington, Seattle,Washington
Received 9/13/05; revised12/20/05; accepted1/26/06.
Grant support: NIH/National Cancer Institute (P50 CA83636) and the Canary
Foundation.
The costs of publicationof this articlewere defrayedinpart by the paymentof page
charges.This article must therefore be hereby marked advertisement in accordance
with18 U.S.C. Section1734 solely toindicatethis fact.
Requests for reprints: Nathalie Scholler, Public Health Sciences/Translational
Outcomes Research Laboratory, Fred Hutchinson Cancer Research Center, 1100
FairviewAvenue North, Mailstop M5-A864, P.O. Box 19024, Seattle,WA 98109-
1024. Phone: 206-667-3170; Fax: 206-667-2537; E-mail: nscholle@fhcrc.org.
F2006 American Associationfor Cancer Research.
doi:10.1158/1078-0432.CCR-05-2007
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and generation (22). Our goal was to develop a cost-effective
research-quality assay for CA125 that would require only a few
microliters of serum and enable us to explore the potential for
multiplexing and/or combining CA125 with novel markers
such as human epididymis protein 4 (HE4; refs. 23, 24) for use
in a CM and a longitudinal algorithm.
Bead-based ELISA assays require relatively small volumes of
sample material (25, 26). The technology derives from
sandwich ELISA assays but uses spectrally discrete polystyrene
beads, or microspheres, instead of plastic surfaces to immobi-
lize the capture antibody. Bio-Rad Laboratories, Inc. (Hercules,
CA) commercializes carboxy-coated microspheres internally
labeled with two fluorescent dyes that produce up to 100
different spectral addresses, and a Multiplex Suspension Array
System (Luminex 110S system) whose reading system (Bio-Plex
reader) functions on the same principles as a flow cytometer.
Similar to a sandwich ELISA assay, each antibody-coupled
microsphere captures antigens that are detected with a
biotinylated antibody and phycoerythrin-conjugated streptavi-
din (SA-PE). For each capture antibody–coupled microsphere,
the Bio-Plex reader simultaneously measures the fluorescent
signals of the microsphere’s particular spectral address and of
the SA-PE. Each mean fluorescence intensity (MFI) reading
corresponds to the average of the fluorescent signals from 100
antibody-coupled microspheres of a particular spectral address;
duplicate measurements are not required. In the absence of
cross-reactivity, each reading can assess the concentration of
multiple markers that are detected by spectrally distinct beads,
further diminishing the sample volume needed.
We developed CA125 bead-based assays using commercially
available antibodies directed against the M11-like or the
OC125-like epitopes, and an HE4 bead-based assay using
the only available antibody pair for HE4 (13, 24). Evaluation of
the candidate bead-based assays was based on several objectives
and criteria. First, laboratory analyses were done to assess
the antibody binding to the beads, the antigen affinity of the
capture antibody after immobilization on the beads, and the
antibody performance as a sandwich pair against known
concentrations of antigen using purified antigen and selected
sera. The top two performing CA125 antibody pairs in the
bead-based assays were then evaluated for multiplexing with
HE4, and the best assays were evaluated in patient sera for their
reproducibility, validity, and performance relative to the
standard CA125II RIA. As the ultimate objective is to be able
to identify ovarian cancer cases early, the bead-based assays
were also evaluated for their temporal stability in serial blood
samples (27). Screening decisions based on CA125 most
commonly use a single-threshold screening rule that prompts
referral for follow up when CA125 values are found above the
reference range, but performance can be improved by use of a
longitudinal algorithm when a marker is stable over time in
women (28).
Materials and Methods
Overall strategy to develop, calibrate, and evaluate the bead-based
assays. In the first phase of the work, we compared the performance
of five anti-CA125 antibody couples (three antibody pairs in only one
orientation and one antibody pair in both orientations) in a bead-
based ELISA platform. All capture antibodies were coupled to the
microspheres at five different concentrations and each coupling was
validated by direct detection with a goat anti-mouse antibody. After
validation, capture antibody-coupled microspheres were incubated
with the control antigen diluted in assay buffer or in pooled control
sera (CA125 < 13 units/mL as measured by CA125II RIA) diluted
4-, 20-, 50-, or 200-fold. The antigen binding was detected with a
biotin-conjugated antibody followed by SA-PE (Bio-Rad Laboratories)
and measured in MFI by the Bio-Plex reader. Antibody pairs that did
well were used to test nonblinded positive and negative control sera
as identified by CA125II RIA and diluted 4- or 20-fold. A bead-based
assay was similarly optimized for HE4, using the only available pair
of capture and detection antibodies. The potential for multiplexing
was explored by combining beads for the HE4 assay with beads for
each of the two best CA125 assays in a single well, and comparing
MFI levels to those obtained without multiplexing for both CA125
and HE4.
In the second phase of the work, the two best pairs of CA125
antibodies and the HE4 antibody pair were analyzed in blinded
patient sera. To assess reproducibility for each of the three antibody
pairs, pooled negative serum and CA125/HE4-positive serum were
measured repeatedly over 10 days. Serum replicates were used to
estimate coefficients of variation (CV). In addition, 204 patient
samples from 191 women with and without ovarian cancer were
assayed twice over the same 10 days; reproducibility was assessed by
the correlation between the two experiments. Results from the first
experiment for each CA125 and HE4 antibody pair were also used to
correlate the findings to the standard CA125II RIA or HE4 ELISA and
to generate receiver operating characteristic (ROC) curves assessing
diagnostic accuracy.
Mouse mAbs. Anti-CA125 mAb were acquired from Fujirebio
Diagnostics (OC-125 and M11), Fitzgerald Industries International,
Inc. (FIII, Concord, MA; M8072320 and M8072321, and M002201 and
M002203), and Research Diagnostics, Inc. (RDI, Flanders, NJ; X306 and
X52). M8072320, M002201 and X306 epitopes are OC-125-like,
whereas M8072321, M002203, and X52 are M11-like. Manufacturers
recommend using M11, X306, and M002201 as capture antibodies. For
the HE4 assay and to test multiplexing, one complementary pair of anti-
HE4 mAbs (3D8 and 2H5) were used (24). Antibodies were dialyzed
against Dulbecco’s PBS (Invitrogen Corporation, Carlsbad, CA) when
needed. Carboxy-coated microspheres (Bio-Rad Laboratories) were
coupled with five concentrations (0.2, 1, 5, 10, and 20 Ag/mL) of the
capture antibodies or with both antibodies of the same pair if no
recommendation was given (FIII M8072320 and M8072321). Detec-
tion antibodies were biotinylated using the EZ-Link-sulfo-NHS-
biotinylation kit (Pierce, Rockford, IL) according to the manufacturer’s
instructions and dialyzed against PBS (Pierce Slide-a-Lyzer, 7 kDa
molecular weight cutoff).
Study population and serum sample collection.
were recruited between 1999 and 2003 to support protocols of the
Pacific Ovarian Cancer Research Consortium by physicians at Pacific
Gynecology Specialists, Swedish Medical Center, the University of
Washington/Seattle Cancer Care Alliance, and Virginia Mason Medical
Center. Cases (n = 66) were defined as having invasive epithelial
carcinoma confirmed by standardized review of medical records and
pathologist examination of paraffin-embedded tissue; histologies
represented clinical practice and included 33 serous, 7 endometrioid,
3 mucinous, 3 clear cell, 6 undifferentiated, and 14 other. International
Federation of Gynecology and Obstetrics stage distribution similarly
reflected clinical practice, including 11 stage I, 5 stage II, 37 stage III, 11
stage IV tumors, and 2 unknown stages. Non-cases, representing
women free of ovarian cancer (n = 125), consisted of surgical patients
(n = 72) and women (n = 53) enrolled in screening trials who were
free of ovarian cancer two or more years after serum collection. Thirteen
of the screening trial participants provided serial blood samples
collected 1 year apart, yielding 66 blood samples for analysis from
the screening population. Participants’ characteristics are reported in
Table 1. In addition, a pool of sera from nine healthy postmenopausal
women collected in a volunteer blood drive was used as negative
Study participants
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control. Each patient provided written informed consent and a medical
records release form approved by the Fred Hutchinson Cancer Research
Center Institutional Review Board.
All blood samples were collected in serum separator tubes (BD
Vacutainer, Becton Dickinson and Company, Franklin Lakes, NJ) and
processed according to the manufacturer’s instructions. Blood was
allowed to coagulate at room temperature for at least 30 minutes but no
longer than 4 hours. The serum was aliquoted and stored at ?80jC
until analysis (3-5 years later). Surgical samples were obtained prior to
surgery at a clinic visit or in the operating room. All serum samples were
characterized for CA125 with the research standard CA125II RIA
(FDI) in duplicate using 0.2 mL of serum as recommended by the
manufacturer to support Pacific Ovarian Cancer Research Consortium
work (16, 24). A subset of the patient specimens (n = 68, including
6 cases and 62 controls) also had CA125 measurements obtained for
a different study using a microparticle enzyme immunoassay (AxSYM
CA125 assay, Abbott Laboratories, Abbott Park, IL). HE4 serum levels
were measured in another subset (n = 106, including 35 cases and 71
controls) using sandwich ELISA for a Pacific Ovarian Cancer Research
Consortium validation study (24).
CA125 and HE4 bead-based immunoassays.
evaluation of the bead-based assays were done in filter plates (Millipore
Corporation, Billerica, MA). A vacuum manifold (Millipore) was used
to drain reagents and wash steps. All incubations were done at room
temperature, in the dark, and on a plate shaker. Carboxy-coated
microspheres (Bio-Rad Laboratories) were resuspended at each step by
ramping the plate shaker to a speed of 1,000 rpm, then reducing to 300
rpm for the incubation. Filter plates were pre-wet with assay buffer prior
to adding the microspheres. Capture antibodies were covalently
coupled to the microspheres using the Amine Coupling Kit (Bio-Rad
Laboratories) according to the manufacturer’s instructions. Briefly,
microsphere stock solutions were dispersed by vortexing and water
bath sonication (Branson ultrasonic cleaner, Danbury, CT), and then
checked visually for aggregation. For each coupling, a 100-AL aliquot of
1.25 ? 106monodispersed microspheres was transferred to a coupling
reaction tube provided in the kit. The microspheres were washed with
the kit bead wash buffer, resuspended in 80 AL of the kit bead activation
buffer, vortexed, and sonicated. Freshly made solutions of 10 AL of
50 mg/mL 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydro-
chloride (Pierce) and N-hydroxy-sulfosuccinimide (Sulfo-NHS, Pierce)
were added sequentially to the coupling tubes to stabilize the reaction
and activate the microspheres. After vortexing, the microspheres were
incubated (rotating) for 20 minutes, and then washed and resuspended.
Various dilutions of capture antibodies were added, the volumes
adjusted to 500 AL and the coupling reaction was incubated for 2 hours.
After one wash, the coupled microspheres were blocked with the kit’s
blocking buffer for 30 minutes. Finally, microspheres were resuspended
in storage buffer, counted using a hemocytometer and stored at 4jC.
To test the coupling efficiency, 5,000 microspheres were incubated for
30 minutes with 2 Ag/mL of biotinylated goat anti-mouse IgG H + L
(Invitrogen) followed by three washes and a 10-minute incubation with
SA-PE. Labeled microspheres were washed thrice, resuspended in 125
Development and
AL of assay buffer, and analyzed with the Bio-Plex reader. MFI of at least
2,000 was considered an efficient coupling. Our criterion to identify the
best pairs of antibodies in the first phase of the work was the greatest
ratio between the MFIs measuring between 0.1 and 1,000 units/mL of
CA125. HE4-Ig fusion recombinant protein was assayed in the same
samples on the same plates, independently, and multiplexed with each
of the CA125 bead-based assays. Criterion for acceptable multiplexing
was MFI consistency in the absence and in the presence of reagents for
another bead-based assay. All assays of patient samples were run using
the same lots of coupled microspheres and biotin-conjugated detection
antibodies.
Statistical analyses. All analyses were done in S-Plus version 6.2
(Insightful, Seattle, WA). Assay reproducibility for the two CA125 bead-
based assays and the HE4 assay was assessed by calculating the CV
among control replicates and estimating Pearson correlations between
repeated experiments. Positive control serum (CA125 level, 231 units/
mL as measured by CA125II RIA; HE4 level, 5 ng/mL as measured by
ELISA) was measured 19 times in 10 separate experiments, providing 19
replicates for each assay. The pool of negative control serum (CA125 < 7
units/mL by RIA; HE4 undetectable by ELISA) was measured 8 times in
triplicate and 11 times alone in 10 separate experiments that included
one or two plates, providing 35 replicates for each assay. Among the
negative and positive serum controls, an ANOVA was used to assess
day-to-day variation and plate variations within days. The criteria for
inclusion of data were that no errors were reported by Bio-Plex. CV’s
were calculated for positive and negative serum controls, on the raw
scale for comparability with published estimates for standard assays. All
other analyses were done after a log transformation was applied to
reduce skewness in the distribution. Pearson correlations were
estimated using all serum specimens for which repeated results were
obtained.
Validity of the CA125 bead-based ELISAs was assessed by correlating
their respective CA125 concentrations to those obtained by the
standard CA125II RIA in the 204 patient serum samples. Pearson
correlation coefficients were calculated overall and within each patient
subgroup (cases and non-cases including surgical patients and
screening participants). Agreement of the HE4 bead-based assay with
traditional ELISA was similarly assessed by Pearson correlation using
only the 106 serum samples for which HE4 ELISA results were available
(24). When a sample was dropped due to errors (n = 6) it was replaced
by its replicate for validity and performance evaluations.
In terms of the diagnostic accuracy of the assays, performance was
assessed by estimation of ROC curves for cases versus all non-cases,
cases versus surgical controls, and cases versus healthy screening
controls in 64 cases and 125 non-cases (55 screening controls and 70
surgical controls) who reported no history of ovarian cancer and for
whom all four assay measurements were available, including the three
bead-based assays (two CA125 and one HE4 assay) and the CA125II
RIA. The ROC curves jointly describe the sensitivity and specificity of
the assay as the threshold for a positive test is allowed to vary. The
area under the curve (AUC; ref. 29) is reported, where 1.0 represents
perfect performance and 0.5 represents what one would expect of
Table1. Participant’s characteristics
n
Age
(median)
Age
(minimum)
Age
(maximum)
CA125II
(median)
CA125II
(minimum)
CA125II
(maximum)
Cases
Non-cases (total)
Surgical
Screening
66
125
70
55
59
55
59
53
37
35
35
38
89
83
83
81
247.26
12.12
13.65
10.25
5.16
3.45
3.45
3.77
2,797.14
1,790.39
1,790.39
190.5
NOTE:The median, minimum, and maximum ages of the participants, and the median, minimum, and maximum CA125 IIRIAvalues byclinicalgroup.
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chance alone. The equality of the AUC’s was tested using a non-
parametric method developed by DeLong et al. (30). ROC curves for
individual bead-based assays were compared with those for the
standard CA125II RIA. In addition, two CMs were constructed by
combining each of the CA125 bead-based assays with the HE4 bead-
based assay using main effects obtained by logistic regression (31); the
AUC for each CM was compared with that of CA125II RIA.
To evaluate the stability over time of the markers using bead-based
assays, the correlation between two measures made in serum samples
collected 1 year apart are reported for each bead-based assay and for the
standard CA125II RIA. The temporal stability of a marker is critical for
theexploitationofalongitudinalalgorithmin ascreeningprogram(28).
Results
Determining optimal conditions for each antibody pair.
Microspheres coupled to serial dilutions of anti-CA125 and
anti-HE4 capture antibodies all surpassed the recommended
MFI of 2,000 in validation tests (data not shown). Assays were
first done with 0.1 to 1,000 units/mL of CA125 antigen purified
from human ascites (FIII) and serially diluted in assay buffer
or in pooled control sera. Figure 1 shows that the optimal
coupling concentration of capture antibodies varied for each
antibody and that the orientation of the FIII antibody
pair M8072320/21 was critical (compare orientation 1 with
2, Fig. 1D). The addition of serum interfered with the
fluorescent signal only when the microspheres were coated
with a low concentration of capture antibody (V1 Ag/mL; data
not shown). Based on a criterion of greatest ratio between the
MFIs measuring 0.1 and 1,000 units/mL of CA125 purified
antigen, we eliminated the FIII pair with clone M8072321 as
capture antibody (orientation 2, Fig. 1D). Beads coupled with
capture antibody concentrations that discriminated best be-
tween various antigen concentrations were selected for further
tests: RDI X306, 5 Ag/mL; FDI M11, 1 Ag/mL; FIII M002201, 1
Ag/mL; and FIII M8072320, 20 Ag/mL.
For assays with human sera, 15 AL of each serum sample
was diluted with 45 AL of human serum diluent (Bio-Rad
Laboratories) and capture antibody–coupled microspheres
were incubated for 30 minutes with 50 AL of the diluted
serum. All four remaining antibody pairs were able to
distinguish CA125-positive and two CA125-negative sera (as
identified by CA125II RIA) at a concentration of 25% (Fig. 2A)
or 5% (data not shown). Because the ratios between positive
and negative sera were higher with the RDI and FIII M8072320/
21 antibody pairs than with the FIII M002201/03 and FDI
M11/OC125 pairs, even at the best serum dilution of 25%, we
focused further efforts on only two CA125 antibody pairs, RDI
and FIII M8072320/21.
To determine the optimal concentration of detection anti-
bodies X52 (RDI) and M8072321 (FIII), coupled microspheres
with the appropriate capture antibody were incubated with four
sera characterized by CA125II RIA and 2-fold serial dilutions of
their corresponding detection antibody (1-8 Ag/mL). The
overall MFI were not significantly modified by the different
concentrations of detection antibodies (data not shown).
The above results were used to determine the experimental
conditions for the rest of the study: the FIII capture antibody
M8072320 was coupled at 20 Ag/mL and the RDI capture anti-
body X306 was coupled at 5 Ag/mL, whereas the detection anti-
bodies were used at 1 Ag/mL for the FIII antibody M8072321
and at 2 Ag/mL for the RDI antibody X52.
Similarly, an HE4 bead-based assay was validated with HE4-
Ig fusion recombinant protein and calibrated with four positive
and negative control sera characterized by sandwich ELISA
test (24). As previously described, beads coupled to 2H5 anti-
HE4 capture antibody were validated with biotinylated goat
anti-mouse IgG (data not shown). Figure 2B shows that
microspheres coupled with 10 Ag/mL of 2H5 and detected
with 6 Ag/mL of biotinylated 3D8 discriminated best between
the positive and negative control sera.
Fig.1. Determinationofoptimalcoupling
concentrations withpurified CA125 antigen
for five pairs ofanti-CA125 antibodies.
Five coupling concentrations of capture
antibody (0.2,1, 5,10, and 20 Ag/mL) were
incubated with serialdilutions of CA125
antigenintheassay diluent.The captured
antigenwas detectedby1 Ag/mLof
biotinylated detectionantibody. A, RDI
antibodies, X306 capture, X52 detection;
B, FDIantibodies, M11capture, OC-125
detection; C and D, FIIIantibodies;
C, M002201capture, M002203 detection;
D, orientation1, M8072320 capture,
M8072321detection; orientation 2,
M8072321capture, M8072320 detection.
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Multiplexing. Multiplexing of CA125 and HE4 bead-based
assays was tested with both the FIII and RDI antibody pairs.
The HE4 MFI readings were not affected by the presence
of the anti-CA125 reagents (Fig. 2C). However, the MFI of
both the RDI (Fig. 2D) and the FIII (data not shown)
CA125 assays increased with the addition of HE4 bead-based
assay components, suggesting the cross-reactivity of the anti-
HE4 antibodies with the anti-CA125 antibodies. Accordingly,
multiplexing was not used for analyses in blinded patient
sera.
Assay reproducibility.The best CA125 antibody pairs (FIII
8072320/21 and RDI X306/X52) and the HE4 antibody pair
were then evaluated in blinded serum from our study
population. All the assays were done with the sera diluted
4-fold, thus, requiring only 0.015 mL of serum per assay per
sample. Bead coupling reactions were highly reproducible as
shown by validation results with a goat anti-mouse antibody to
quantify the mouse antibody bound to the beads (MFI < 8% SD
for eight couplings done over 6 months).
Reproducibility was assessed using multiple replicates of
negative (n = 35) and positive (n = 19) serum controls to
estimate a CV for each assay. Plate effects assessed by ANOVA
included small day-to-day variations found in both sera
controls for the CA125 RDI and HE4 assays, and small order
effects found both in the positive controls for the CA125 FIII
and HE4 assays and in the negative controls for the CA125 RDI
and HE4 assays (data not shown). The average CVs for the
CA125 RDI and FIII bead-based assays were, respectively, 18.2
and 15.7 for the pooled control sera, and 18.9 and 10.2 for the
CA125/HE4-positive sera. The CVs for the HE4 assay were
higher, ranging from 26.6 for the positive sera to 32.6 for the
pool of negative sera.
For further assessment of reproducibility, blinded samples
from191 patients were tested twice in independent experi-
ments conducted identically 1 week apart. Overall, the Pearson
correlations for replicates were 0.99, 0.95, and 0.95 for the
RDI, FIII, and HE4 assays, respectively (Table 2). The estimated
correlations within subgroups (i.e., cases, surgical controls, and
screening controls) ranged from 0.64 (FIII screening controls)
to 1.00 (RDI cases) providing strong evidence of assay
reproducibility, even within the narrower range of antigen
levels characteristic of healthy controls. The RDI assay yielded
modestly higher correlations between replicates than the FIII
assay overall and in all three subgroups. Reproducibility for
the HE4 bead-based assay was similar, with correlations
slightly higher in screening controls (0.86 versus 0.83 and
0.64) but lower in surgical controls (0.90 versus 0.98 and 0.97;
Table 2).
Fig. 2. Calibrationand multiplexing of thebead-basedassays. A, four sera with known CA125 concentrationlevels were diluted 4-foldandused to compare four CA125
bead-basedassays. Alldetectionantibodies wereused at1 Ag/mL.The capture antibodies were immobilizedonmicrospheres at 5 Ag/mL for RDImAb, 20 Ag/mL for FIII
M8072320mAb,1 Ag/mLforFDImAb, and1 Ag/mLforFIIIM002201mAb. B, microspheres were coupledwithfive concentrationsof 2H5 anti-HE4capturemAb as shown,
andincubatedwith two HE4-positive sera (pos1and pos 2; HE4 concentrations of 5 ng/mL as determinedby ELISA) and two HE4 negative sera (neg1and neg2; HE4
concentrations nondetectable by ELISA) diluted 4-foldinassay buffer. HE4 serumlevels were detected with 6 Ag/mL of 3D8 anti-HE4 biotin-conjugated mAb. C, optimized
HE4 bead-based assay was testedinthe absence orinthe presence of CA125 bead-basedassay reagents and/orinpresence of serialdilutions of CA125 purifiedantigen
inassay diluent (0, 30, 300, and 3,000 units/mL). D, CA125 bead-basedassayusing RDIantibodies was testedinthe absence orinthe presence of HE4 bead-basedassay
reagents and/orinpresence of serial dilutions of HE4-Igantigen (0, 0.5,1, and 2 Ag/mL).
Bead-Based Assays for Ovarian Cancer MarkerValidation
www.aacrjournals.orgClin Cancer Res 2006;12(7) April1, 20062121