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Journal of Clinical Microbiology, May 2000, p. 1823-1826, Vol. 38, No. 5
0095-1137/00/$04.00+0
Standardization of Immunoglobulin M Capture
Enzyme-Linked Immunosorbent Assays for Routine Diagnosis of
Arboviral Infections
Denise A.
Martin,*
David A.
Muth,
Teresa
Brown,
Alison J.
Johnson,
Nick
Karabatsos, and
John T.
Roehrig
Division of Vector-Borne Infectious Diseases,
National Center for Infectious Diseases, Centers for Disease
Control and Prevention, Public Health Service, U.S. Department of
Health and Human Services, Fort Collins, Colorado 80522
Received 3 August 1999/Returned for modification 1 November
1999/Accepted 8 March 2000
 |
ABSTRACT |
Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay
(MAC-ELISA) is a rapid and versatile diagnostic method that readily
permits the combination of multiple assays. Test consolidation is
especially important for arthropod-borne viruses (arboviruses) which
belong to at least three virus families: the Togaviridae,
Flaviviridae, and Bunyaviridae. Using prototype
viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus
family. Furthermore, by grouping antigens geographically and utilizing
known serological cross-reactivities, we have reduced the number of
antigens necessary for testing, while maintaining adequate detection
sensitivity. We have determined that a 1:400 serum dilution is most
appropriate for screening antiviral antibody, using a
positive-to-negative ratio of 
2.0 as a positive cutoff value. With a
blind-coded human serum panel, this combined MAC-ELISA was shown to
have test sensitivity and specificity that correlated well with those
of other serological techniques.
| |
INTRODUCTION |
Most medically important
arthropod-borne viruses (arboviruses) belong to three virus families:
the Togaviridae, Flaviviridae, and
Bunyaviridae. Many of these viruses are considered emerging or reemerging infectious diseases that can be readily transported from
one area of the world to another. Because of the wide variety of
viruses, arboviral diagnostic serology is complex. A recent survey of
diagnostic laboratories in the United States that perform arboviral
testing determined that the indirect immunofluorescence assay
(2), plaque-reduction neutralization test (PRNT) (2, 17), hemagglutination-inhibition test (10), and
complement fixation test (8) were still widely used (M. Bunning, personal communication). Many of these tests are technically
demanding, making them difficult to apply reproducibly, and are often
poor measures of the early antibody immunoglobulin M (IgM).
Furthermore, obtaining results from these tests may take several days
and require paired serum samples or live cell culture. Virus isolation
is rarely a viable option even in epidemic situations, due to poorly timed specimens.
The IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA)
was designed specifically to detect IgM antibody, which is a valuable
tool for rapid diagnosis of acute viral infections. IgM appears early
in infection, rises rapidly in the disease course, and is usually less
virus cross-reactive than IgG (16). While many separate IgM
ELISAs have been developed for arboviruses, these tests are not well
standardized (1, 13, 15, 20). Most use a commercial source
of anti-human IgM as capture antibody, but they also use purified virus
as antigen, which is impractical for multiple agents (4-7, 11,
14). Furthermore, the selection of the screening serum dilution
has not been extensively evaluated (7, 19) but has relied on
results from an earlier study (21). These assays use
polyclonal antiviral antibody as a detector, which is impractical for
all but the largest reference laboratories that prepare these
specialized reagents routinely (4-7, 11, 14). These
polyclonal antibody detectors vary in batch-to-batch potency and can be
quite virus cross-reactive, which limits test specificity. Finally, the
criteria that indicate positive reactions, usually expressed as a
positive-to-negative (P/N) ratio, have been inconsistent.
We have developed, implemented, and validated a standardized MAC-ELISA
for rapid screening of human serum samples for various arboviruses.
These new tests use a defined set of antigens, tailored to the
geographic origin of the specimen. The method is readily standardizable, using a commercial source of anti-human IgM capture antibody and broadly reactive antiviral monoclonal antibody
(MAb)-enzyme conjugates as detectors. This approach has resulted in a
reliable, rapid, and readily transferable system for monitoring
arbovirus disease.
| |
MATERIALS AND METHODS |
Human serum.
Serum specimens were obtained from the
Centers for Disease Control and Prevention, Division of
Vector-Borne Infectious Diseases (DVBID), serum bank, which
consists of specimens sent to DVBID for arboviral diagnostic testing.
Sera were selected on the basis of a positive result to either eastern
equine encephalitis virus (EEE), St. Louis encephalitis virus (SLE), or
La Crosse encephalitis virus (LAC) in a previously performed serologic
test. For the MAC-ELISA standardization, 22 positive and 13 negative
sera were used for EEE, 24 positive and 12 negative sera were used for
SLE, and 18 positive and 9 negative sera were used for LAC. Positive serum specimens with high, medium, and low reactivities were chosen.
PRNT.
The serum dilution PRNT was performed with Vero cells,
as previously described (2). The following viruses were used
to represent the three viral genera in all tests: EEE strain NJ/60, SLE
strain TBH-28, and LAC strain Original. Endpoints were determined at a
90% plaque-reduction level.
MAC-ELISA.
This test was a modification of the assay
previously reported by Beaty et al. (2). Goat anti-human IgM
(PerImmune, Inc., Rockville, Md.) was used as capture antibody, and
aliquots were stored at 
70°C long-term, thawed once, and held at
4°C thereafter. Carbonate-bicarbonate buffer (0.015 M sodium
carbonate, 0.035 M sodium bicarbonate, pH 9.6) was used as coating
buffer. Coated plates (Immulon II 96-well microtiter plates; Dynatech
Industries, Inc.) were incubated overnight at 4°C in a humidified
container and were stable for 2 weeks. A blocking step using
phosphate-buffered saline with 0.5% Tween 20 and 5% nonfat dry milk
was added to reduce overall background and increase test sensitivity.
Blocking and coating buffers and undiluted conjugates after
reconstitution were stored at 4°C. A standard five washes with a
Skatron microplate washer (Skatron Instruments, Sterling, Va.) were
used except before the addition of substrate where 10 washes reduced
overall background. Sera and homologous positive and negative antibody
controls were titrated using 10 twofold dilutions starting at 1:100 to
evaluate the optimum screening dilution. The diluent used for test sera and antigens was phosphate-buffered saline with 0.5% Tween 20 without
fetal bovine serum as recommended by Beaty et al. (2). Diluted antibody was stable for only 7 to 10 days and was discarded thereafter (personal observation). Viral and normal antigens, prepared
as sucrose-acetone extracts of infected suckling mouse brains
(2), were obtained from the DVBID reference collection. Reconstituted undiluted antigen was stored at 20°C, and antigen dilutions were used once and discarded. An overnight incubation at
4°C was used for the antigen step to increase test sensitivity, after
observing a decrease in measured absorbance
(A450) values when using a 2-h antigen
incubation. Group-reactive MAbs were purified and conjugated to
horseradish peroxidase by Jackson Immunological Laboratories, Inc.
(West Grove, Pa.), and used as detector antibodies. The following MAbs
were used: 2A2C-3 for alphaviruses (15), 10G5.4 for
California group viruses (a gift from George Ludwig via Barbara Israel,
University of Wisconsin) (18), and 6B6C-1 for flaviviruses
(22). Use of MAb detectors eliminated the necessity for a
secondary antibody addition. Commercially prepared
3,3',5,5'-tetramethylbenzidine (TMB-ELISA; GIBCO Bethesda Research
Laboratories, Inc., Gaithersburg, Md.) was used, and the substrate
reaction was stopped with 1 N sulfuric acid. Reactions were measured
using a Bio-Rad microplate reader (Bio-Rad Laboratories, Hercules,
Calif.) at an absorbance of 450 nm.
All reagents were titrated individually by using a twofold dilution
series that provided reagent excess in the first well of the series.
Reagent dilutions which resulted in optical densities between 0.8 and
1.0 were chosen.
Calculation of P/N values.
Calculations that were performed
followed guidelines set in the work of Beaty et al. (2) with
the following modifications. The negative serum control P/N must be
<2.0 and the positive serum control P/N must be 2.0 for the test to
be valid. A positive test result was obtained when the P/N of the test
serum was >2.0 and the mean of the A450 values
of the test serum reacted on viral antigen was at least twice the mean
of the A450 values of serum reacted on normal
mouse brain antigen. When the latter criterion was not met due to
nonspecific reaction with the normal mouse brain antigen, the result
was reported as uninterpretable.
Validation testing panels.
Seven positive sera from each
virus group and seven negative sera (confirmed by MAC-ELISA and PRNT
results) were selected, blind coded, and tested in triplicate by
MAC-ELISA by using the 1:400 serum screening dilution. For viruses in
the antigen batteries other than EEE, SLE, and LAC, at least three
positive serum specimens were collected and analyzed using the
screening MAC-ELISA.
| |
RESULTS |
Determination of antigen panels.
We performed a historical
analysis of arboviral testing results produced in our laboratory over
the past 20 years. The vast majority of positive serum samples were
representative of a few viruses that could be localized geographically.
Although it has been shown previously that IgM is less cross-reactive
than IgG (especially for alphaviruses) (5, 23), enough
cross-reactivity exists especially in the family
Flaviviridae (3, 19) and the California serogroup
viruses (1, 7) to permit the establishment of seven
geographically based testing panels representing the medically
important arboviruses. These panels are shown in Table 1. Using these batteries to screen
specimens ensured the detection of virtually all the endemic
arboviruses in that area of the world for those virus families, which
reduced the amount of testing needed for diagnosis, saving time and
resources.
Determination of the MAC-ELISA screening dilution.
Titration
curves for the representative antiviral serum samples are shown in Fig.
1. The resultant curves were typical of ELISA endpoint titrations, which correlated well with concurrently run
PRNT for the same sera. Figure 1 demonstrates the flat line produced by
negative serum specimens even though in some cases the
A450 values were sufficient for negative P/N
values to be in the range of 2.0 to 2.5. To determine the optimum
dilution for screening sera, P/N values for these sera at 1:100, 1:400, 1:1,600, and 1:25,600 dilutions were compared to endpoint titers. Using
chi-square analysis for all three viruses tested, a 1:400 screening
dilution generated P/N ratios in the positive range that correlated
well with measured endpoint titers. Only 2 of 20 positive EEE antibody
specimens were positive by endpoint but negative by 1:400 dilution
screening. One of 12 positive LAC antibody specimens was positive by
1:400 dilution screening and negative by endpoint titration. The 22 specimens testing positive to SLE in the screening MAC-ELISA were also
positive by endpoint titration. All antibody-negative control specimens
were negative by both testing procedures for all three viruses.
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|
FIG. 1.
MAC-ELISA titration curves. Log10 serum
dilution versus A450 is presented.
Representative specimens of high-titered sera ( ), medium-titered
sera ( ), low-titered sera ( ), and negative sera ( ) are
plotted.
|
|
Test validation.
Results of the blind-coded serum testing are
shown in Table 2. MAC-ELISA screening of
sera testing positive to other members of the antigen batteries showed
uniform positive reactions to homologous antigens. Little
cross-reaction was observed within the alphaviruses; however,
Chikungunya virus cross-reacted with o'nyong-nyong virus. Flaviviruses
and California serogroup viruses exhibited extensive cross-reaction
between members of each respective group.
| |
DISCUSSION |
An array of antibody types and subclasses is produced by a normal
host humoral response to viral infection. IgM antibody is produced
early in the immune response (16). The MAC-ELISA
specifically detects IgM, allowing for timely diagnosis of disease. The
capture format of the MAC-ELISA eliminates potential background caused by extraneous antibody, resulting in less-frequent nonspecific reactions and removing false-positive reactions caused by rheumatoid factor (12). Competition between IgM and IgG for antigen
binding is minimized, reducing the occurrence of false-negative
results. Ensuring exact incubation times, especially in the critical
substrate step, yields results that can be compared between tests. The
use of broadly group-reactive MAb conjugates in combination with
virus-specific antigens creates a system in which antibody to many
arboviruses within a genus can be screened for concurrently by using a
single virus-adaptable procedure.
Correlation of the MAC-ELISA results with the PRNT was good. The
occasional disparity that was noted between results of the two tests is
probably explained by the fact that the antibody types detected can be
different. IgM produced early in infection does not always possess
neutralizing activity (7). When the worldwide antibody
panels based on the various exploitable cross-reactions were used
(1, 7, 17, 19), a single MAC-ELISA format incorporating
various antigens gave a rapid and precise picture of the IgM antibody
status of a given serum. Newly emerging arboviruses and those in other
virus families can be added to any battery as soon as the proper viral
antigens have been developed and positive controls are obtained. Using
the MAC-ELISA in tandem with an IgG ELISA (14a) yields
antibody profiles capable of identifying recent infections by separate
measurement of IgM and IgG.
Interpretation of MAC-ELISA results is based primarily on the timing of
the sample and confirmation of those results in another test. A
potentially IgM-positive acute-phase serum sample is defined for
purposes of this test as serum taken at least 8 days and up to 45 days
after onset of symptoms (unpublished data). A positive result by
MAC-ELISA in a single acute-phase specimen is presumptive evidence of a
recent infection with that arbovirus (9), exploiting the
early rising and rapidly declining nature of IgM antibody. The
detection of IgM antibody to a particular arbovirus in a cerebrospinal fluid (CSF) specimen is evidence of infection with that virus (2,
9). However, a negative MAC-ELISA result for a very acute-phase
specimen (day 0 to 7) may reflect an insufficient antibody response
very early in infection rather than no infection. Moreover, MAC-ELISA
results in an acute specimen that remain unconfirmed by PRNT may
reflect the lack of neutralizing ability of IgM produced early in
infection and should not be confused with a false-positive result
(7). Routinely requesting a convalescent-phase specimen in
this situation alleviates this dilemma. In the absence of a convalescent-phase specimen, PCR can be used for confirmation.
Our results showed that the screening dilution (1:400) had a good
correlation with true endpoint values, therefore eliminating the need
for endpoint titrations except for confirmation of positive serum
detected by the initial screening MAC-ELISA. Occasionally, CSF
specimens are submitted, and these are screened undiluted. Using a
screening dilution of 1:400 eliminates most false positives. However,
P/N values in the range of 2.0 to 3.0 have occasionally been determined
to be false positives, requiring endpoint titrations to confirm the
positive reaction (personal observation). In practice, we do MAC-ELISA
endpoint titrations on serum specimens positive in our screening
MAC-ELISA. False-positive sera yielding an endpoint generally graph as
a flat line and demonstrate a fairly stable P/N range, usually under
3.0. However, only a portion of these sera with low P/N values will
fail a confirmatory neutralization test.
Development of this adaptable and rapid MAC-ELISA for detection of
medically important arboviruses in serum and CSF has made clinical
diagnosis more efficient. During the West Nile encephalitis epidemic in
New York City in the summer of 1999, the MAC-ELISA was a key tool in
early detection of cases. The use of SLE virus in the U.S. antigen
panel allowed us to detect infection with WN in this new setting. This
further demonstrated the versatility of virus panels, while making the
process of arbovirus detection streamlined and more efficient.
Nevertheless, the use of more traditional testing methods for some
arboviruses that are not members of the families addressed here must
continue. Use of the MAC-ELISA and IgG ELISA in tandem will allow us to
examine the nature of antibody response in arboviral infections in more detail.
| |
ACKNOWLEDGMENTS |
We thank Alan Dupuis and Rebecca Deavours for their help with the
preparation of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Vector-Borne Infectious Diseases, National Center for Infectious
Diseases, Centers for Disease Control and Prevention, P.O. Box 2087, Fort Collins, CO 80522. Phone: (970) 221-6445. Fax: (970) 221-6476. E-mail: DZM9{at}CDC.GOV.
| |
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Journal of Clinical Microbiology, May 2000, p. 1823-1826, Vol. 38, No. 5
0095-1137/00/$04.00+0
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Abstract
Introduction
