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Clinical and Diagnostic Laboratory Immunology, November 2000, p. 915-919, Vol. 7, No. 6
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Combined Cell Culture Enzyme-Linked Immunosorbent
Assay for Quantification of Poliovirus Neutralization- Relevant
Antibodies
A. F.
Wahby*
Department of Molecular Biology, National Research Center, Dokki,
Cairo, Egypt
Received 28 April 2000/Returned for modification 23 June
2000/Accepted 25 August 2000
 |
ABSTRACT |
A combined cell culture enzyme-linked immunosorbent assay
(CCC-ELISA) was developed for measuring the neutralizing antipoliovirus antibodies in human sera. The binding of different concentrations of
each of the three poliovirus types to BGM cells in the presence and
absence of a constant dilution from each test and reference serum was
measured in the CCC-ELISA. The titers of the viruses neutralized by
each serum were measured with the titration curves and used for
interpretation of neutralizing titers to the three poliovirus types.
Analysis of human sera revealed that the sensitivity and specificity of
the CCC-ELISA and the microneutralization assay were comparable. The
CCC-ELISA is nonsubjective, rapid, and highly reproducible.
Furthermore, the CCC-ELISA could potentially be used as a
seroepidemiologic tool for assessment of the humoral response to the
cell culture infectious viruses.
| |
INTRODUCTION |
Poliovirus is a picornavirus of the
Enterovirus genus that infects susceptible cells through a
specific receptor which binds the virus and changes its conformation
(1). Sera from subjects infected or vaccinated with
poliovirus contain antibodies to different functional and structural
proteins of the virions (4). Polioviruses can be divided
into three types on the basis of their neutralization reactions with
specific immune sera. Four main antigenic sites have been identified in
each of the three poliovirus types (14). Three major
neutralization antigenic sites have been mapped to three major
polypeptides (VP1, VP2, and VP3) composing the virion surface (11,
15, 17). Detection of the neutralizing antiviral antibodies forms
the basis for evaluation of protection. Neutralization of the
poliovirus by the antiviral antibodies is believed to involve different
mechanisms which block one or more of the viral functions of attachment
to cells, penetration, and uncoating (2, 3, 16) or to occur
by postadsorption neutralization (13, 18).
In vitro virus neutralization is the main standard assay for the
detection and measurement of the neutralizing poliovirus antibodies.
Although the polioviruses induce clear visible cytopathic effects (CPE)
in susceptible cell cultures, the interpretation of neutralizing titers
is influenced by the subjective visual scoring of CPE and the long time
required for low virus titers to develop CPE. For these reasons, a
number of assays have been described and evaluated as alternatives to
the standard assay.
Different nonfunctional assays which avoid the use of cell cultures and
living viruses have also been described (5, 6, 7, 9, 10).
Although these nonfunctional assays are relatively rapid and less
tedious than virus neutralization, they have not replaced
neutralization assays for the measurement of poliovirus antibodies.
This is mainly because of the low degree of correlation between
functional and nonfunctional tests at low antibody concentrations (19, 21), in addition to the fact that nonfunctional tests measure all or some of the neutralizing antibodies, as well as other
types of antibody molecules, and require specific monoclonal antibodies. Another sensitive procedure which avoids the visual scoring
of CPE but requires special equipment and radioactive isotopes was also
described previously (12).
In this article, a relatively rapid and nonsubjective assay for the
detection of neutralizing antiviral antibodies is described. The assay
was based on establishing an enzyme-linked immunosorbent assay (ELISA)
for measuring cell-associated viruses in the presence and absence of a
neutralizing antiserum. The assay has been named the combined cell
culture (CCC)-ELISA. Also, an evaluation of the CCC-ELISA for the
assessment of humoral responses to poliovirus is presented.
| |
MATERIALS AND METHODS |
Virus strains.
The poliovirus strains were Sabin types 1, 2, and 3 grown in BGM cell cultures. Virus harvests in tissue culture
medium 199 (TCM) were cleared from cell debris by centrifugation for 15 min at 104 × g. The supernatants were
distributed into small aliquots and stored at 
70°C.
Antisera.
For CCC-ELISA, human serum samples positive for
polio (positive human sera [PHS]) were obtained from a previous study
for detection of neutralizing antibodies. Fifty human sera were
obtained from infants between the ages of 1 and 2 years admitted to
local hospitals for reasons other than infectious diseases. The sera were stored frozen at 20°C and heat inactivated before use.
Informed consent to use the serum for the study was obtained from each individual or his or her guardians.
Cell cultures.
BGM cells, a continuous cell line of African
monkey kidney origin from the American Type Culture Collection, were
used for poliovirus propagation and titration. After trypsinization,
the BGM cells were suspended in Dulbecco's modified Eagle's medium supplemented with 10% inactivated fetal bovine serum (FBS).
Thereafter, 100-µl volumes of BGM cell suspension (105
cells per ml) were dispensed in 96-well tissue culture plates and
incubated at 37°C in a CO2 incubator. After overnight
incubation, the formed monolayers were washed with TCM and used for
performing the titrations in the microneutralization assay (NT) and
CCC-ELISA. TCM supplemented with either 1% FBS (TCM-1%) or 5% FBS
(TCM-5%) was used as a maintenance medium for cell-virus cultures.
TCM-1% was used as a dilution buffer for the sera and viruses.
NT.
The NT was performed according to a standard assay
(20), with minor modifications. Test sera were used to
prepare twofold dilutions in 96-well transfer plates (Dynatech
Laboratories), and a 25-µl volume of each dilution was added to 3 wells. A 25-µl volume containing 75 to 100 50% tissue culture
infectious doses (TCID50) of virus was added to each well,
and the antigen-antibody mixtures were incubated at 37°C for 2 h. Thereafter, the mixtures were inoculated onto preformed monolayers
of BGM cells in a culture plate, and 100 µl of TCM-5% was added to
each. The cultures were incubated for 7 days at 37°C in a
CO2 incubator and observed periodically for the development
of CPE. Titers were determined from serum dilutions in triplicate by
the Spearman-Karber procedure (8).
CCC-ELISA.
Serial 10-fold dilutions of each poliovirus type
were prepared in transfer plates. A 100-µl volume of each virus
dilution was mixed with either 100 µl of a constant serum dilution
(neutralization plot) or 100 µl of TCM-1% (virus control plot). Each
serum was mixed with an equal volume of TCM-1% for use as a control
(serum control plot). The reaction mixtures were incubated overnight at
4°C, and 50 µl of each reaction mixture was transferred to three
wells of preformed BGM monolayers. TCM-1% was added to the cell-virus
cultures (100 µl/well), and the cultures were incubated overnight at
37°C in a CO2 incubator.
For detection of the cell-associated viruses, the cultures were washed
with phosphate-buffered saline (pH 7.4) (PBS) and incubated with the
appropriate indicator antibody against the reference serum for the
poliovirus type (100 µl/well) for 2 h at 4°C. The cells were
washed with PBS, and goat anti-human polyvalent immunoglobulin (G, A,
and M) conjugated with peroxidase (Sigma Chemicals, St. Louis, Mo.) was
added (100 µl/well) at a dilution of 1:1,000. Plates were incubated
for 1 h at 4°C and washed with PBS. The enzyme substrate (0.04%
O-phenylenediamine HCl-12% H2O2 in
citrate buffer [pH 5] was added (200 µl/well) and incubated for
1 h at 37°C. Thereafter, 150 µl of the supernatant from each
well was transferred to an ELISA plate preloaded with 2 M
H2SO4 (50 µl/well), and the absorbance was
measured at 490 nm.
The titration curves were established by plotting the log of the virus
dilutions versus the ELISA optical densities (OD) using
a polynomial
fitting. The log of the virus dilutions at the intercepts
of the serum
control plot with the virus neutralization plot and
the virus control
plot were taken as the endpoints. The difference
between the endpoints
for a virus titration in the presence and
absence of a serum was
designated
V. The virus neutralized by
a constant dilution of a
serum was the antilog of
V. The neutralization
titers were
interpreted as relative titers by parallel titration
of a reference
serum.
Statistical analysis.
Simple regression analysis was used to
compute the correlation coefficient (r) between the
neutralization titers determined by the NT and the CCC-ELISA (Apple
computer software). A probability value of <0.05 was considered significant.
| |
RESULTS |
Establishment of the CCC-ELISA.
Ten PHS were screened for
antipoliovirus antibodies in the standard NT. Sera with high
neutralizing titers were chosen as reference antisera RHS1, RHS2, and
RHS3 for serotypes 1, 2, and 3, respectively (Table
1). RHS1 and RHS2 were polyspecific,
whereas RHS3 was type specific.
During the process of developing and optimizing the assay conditions,
the influence of several factors on the titration curves,
and
consequently on the CCC-ELISA results, was investigated. The
cell-associated viruses were better recognized by the corresponding
reference serum, and lower OD values were obtained when a pool
of the
reference antisera was used for the 24-h time point (Fig.
1). The neutralization plots seemed to be
of sigmoid shape, so
fitting of the linear part of the plot is
theoretically the most
appropriate method for analyzing the data.
Because it is practically
difficult to identify the linear part of the
neutralization curve,
the smooth and polynomial fitting modes were
tried for fitting
of the titration curves. The polynomial fitting was
the most practical
under the experimental conditions used, and the mean
value of
r for fitting 10 titration curves was 0.98, with a
standard deviation
of <0.01. The titration curves established for
cell-associated
viruses at 90 min and 24 h were of different
shapes (Fig.
1).
The slope of the titration curves at the endpoints
increases by
increasing the period of cell-virus incubation, which
increases
the sensitivity in measuring the endpoints for virus
titration.
However, a decrease in the stability of the cultures was
noticed
with prolonged incubations and resulted in a less clear
endpoint.
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FIG. 1.
Recognition of cell-associated virus at different time
points from the start of incubation of poliovirus type 2 with BGM
cells, using two indicator antibodies. Values were plotted at 90 min
( ) and 24 h ( ) using RHS2 and at 90 min ( ) and 24 h
( ) using a pool of the reference sera. The RHS2 control plot was
established at 24 h ().
|
|
In the standard CCC-ELISA, a constant dilution of the reference serum
possessing a titer of 4 to 10 in the NT was used for
establishing the
virus neutralization curve and as the indicator
antibody. Serial
(10-fold) dilutions of each poliovirus type initially
containing
~10
4 to 10
9 TCID
50 were enough to
perform the titration when overnight incubation
periods were used for
measuring the cell-associated virus. The
dilutions recommended by the
manufacturer for using the horseradish
peroxidase-anti-human
immunoglobulin conjugate in the conventional
ELISA (1/1,000 or 1/2,000)
were found to be appropriate for the
CCC-ELISA.
Specificity, reproducibility, and sensitivity of the
CCC-ELISA.
To check the specificity of the CCC-ELISA, the
neutralization curves for comparable levels of the three poliovirus
types were established with the monospecific reference serum RHS3 (Fig.
2). The neutralization curves
demonstrated that the titers of poliovirus types 1 and 2 were close,
whereas the titer of type 3 was much lower. Similar results were
obtained with the polyspecific antisera RHS1 and RHS2, with the ranking
of the neutralizing antibody titers to the three viruses and the
observed magnitude of neutralization being exactly the same.
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FIG. 2.
Neutralization of poliovirus types 1 (), 2 (), and
3 () with RHS1 (1/1,000), RHS2 (1/100), and RHS3 (1/100) in the
equilibrated serum-virus mixtures and as indicator antibodies. The
initial concentrations of polioviruses used were  104
TCID50 for RHS1 and RHS3 and 106
TCID50 for RHS2. A serum control plot was established in
the absence of virus ().
|
|
The reproducibility of virus and serum control plots in repeat
experiments was checked with a constant set of reagents (Fig.
3). The variation in the endpoints for
five repeat experiments
was negligible, and the estimated virus titer
in five repeated
experiments was 10
7.87 ± 0.025
CCC-ELISA units (mean ± standard deviation), indicating that
the
plots were highly reproducible.
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FIG. 3.
The established virus and serum control plots with
poliovirus type 2 and the corresponding RHS2 in two separate
experiments. , virus control, experiment 1;  , serum control,
experiment 1; , virus control, experiment 2; , serum control,
experiment 2.
|
|
The established neutralization and virus control curves with serum were
parallel over a wide range of serum dilutions possessing
a titer of
10
0.6 to 10
3 in the NT. Neutralization curves
of different shapes were obtained
with the different test sera (Fig.
4) and when different sera
were used as
indicator antibodies. The background for the serum
control was
concentration dependent, and a lower background was
obtained with the
more diluted sera. However, the magnitude of
V decreases by
increasing the serum dilution, which decreases
the accuracy and
sensitivity of the measurements. Furthermore,
false negative predictive
values were obtained at high serum dilutions.
Practically, constant
CCC-ELISA neutralization titers were obtained
with different indicator
antibodies when the test and the reference
sera were titrated in
parallel in repeated experiments. The mean
relative titer ± the
standard deviation for a serum (positive
serum in Fig.
4) in five
repeated experiments was 0.324 ± 0.015.
Levels of neutralizing
antibodies to any of the three serotypes
equivalent to a titer as low
as 0.2 in the NT were detected under
the stated experimental
conditions. Detection of lower levels
of neutralizing antibodies was
not attempted.
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FIG. 4.
Typical titration curves for detection of neutralizing
antibodies according to the standard CCC-ELISA. The virus control ()
and neutralization plots were established with poliovirus type 1 (initial titer of 109 TCID50) and RHS1. The
values of V for the reference (), negative (), and positive
() sera at a dilution of 1:1,000 were 1.71, 0.55, and 0.5, respectively. A serum control plot was established in the absence of
virus ().
|
|
Comparison of the two assays.
The specificity of the assay was
further investigated by comparison of the poliovirus type-specific
antibodies obtained by the NT and the CCC-ELISA. The data for PHS (Fig.
5) and infants (Fig.
6) are summarized in Table
2. The data revealed a high correlation
for each of the three serotypes over the entire range of antibody
concentrations (P < 0.01) for all the comparisons. The
data for the sera negative in the NT were excluded when the coefficient
of correlation was calculated.
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FIG. 5.
Correlation between neutralization titers for poliovirus
types 1 (), 2 (), and 3 () obtained with standard NT and
CCC-ELISA for 10 PHS.
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FIG. 6.
Correlation between neutralization titers for poliovirus
types 1, 2, and 3 obtained with standard NT and CCC-ELISA for 50 infant
sera.
|
|
Analyses of 10 PHS revealed that the numbers of sera recognized as
positive by both the NT and the CCC-ELISA were 9 of 10,
9 of 10, and 7 of 10 for serotypes 1, 2, and 3, respectively.
One serum was negative
for serotypes 1 and 2. Two sera of negative
predictive value in the
serotype 3 NT gave positive signals in
the CCC-ELISA.
Analyses of the infant sera revealed that the numbers of sera
recognized as positive by both the NT and the CCC-ELISA were
46 of 50, 46 of 50, and 46 of 50 for serotypes 1, 2, and 3, respectively.
Three
sera of negative predictive value in the serotype 3 NT gave
positive
signals in the CCC-ELISA.
| |
DISCUSSION |
A new procedure for the detection of neutralizing poliovirus
antibodies is described. In the first step, equilibrated mixtures containing constant amounts of a test serum and various poliovirus concentrations were prepared. In the second step, quantitative parameters for the infectivity of the equilibrated mixtures in BGM cell
cultures were measured in a modified ELISA. The virus titers in the
presence and absence of sera were depicted for the determined
parameters and used to interpret the neutralized virus quantities.
Comparison of the neutralized viruses by reference and test sera
allowed accurate determination of the levels of neutralizing
antibodies. This new type of neutralization assay differs from the NT
in two main aspects. First, virus neutralization was measured at a
constant concentration of serum. Second, the CCC-ELISA results are
based on a nonsubjective estimation of the residual cytopathic virus.
Therefore, several factors which could potentially influence the
sensitivity and reproducibility of the CCC-ELISA neutralization titers,
including the cell-virus incubation period, the ELISA setup, and the
fitting procedure, were investigated.
Although the cell-associated virus could be measured as early as 90 min
after the start of the cell-virus incubation, incubation for 24 h
allowed for the detection of low virus concentrations. In addition, it
seems that some of the cell-associated viruses were not able to
replicate, as described previously (13, 18). The
cell-associated poliovirus could be well recognized by the heterotypic
antibodies, as demonstrated by neutralization profiles for the three
poliovirus types by a monospecific serum in the serotype 3 CCC-ELISA.
Since human sera contained different formulas of antibodies to the
three-poliovirus types, both the serotype-specific and the heterotypic
antibodies could influence each virus neutralization profile. In
agreement with this observation, parallel titration curves were
obtained when dilutions of a single serum were titrated. A reproducible
titer was interpreted from the different neutralization profiles
developed by different indicator antibodies for a virus titration.
However, the assumption that the heterotypic antibodies do not affect
the amount of the neutralized virus was not strictly tested.
One could simply state that the working neutralization mechanisms in
both the standard NT and the CCC-ELISA are mainly the same. However,
because the endpoint is depicted in a plot for reaction mixtures
containing excess virus, it is speculated that some of the proposed
interactions (2, 16) are more favorable for each of the
assays. Although the amount of neutralized virus in each of the
equilibrated mixtures is constant, its determination is always
influenced by the setup of the probing assay. Therefore, to avoid such
variations, a reference serum was always titrated in parallel with the
test sera (19).
Under the conditions used in our experiments, the reproducibility of
the CCC-ELISA titers of the neutralizing antibodies was excellent both
within and between assays. The use of precisely known virus titers and
the reproducible determination of the virus titers were not needed. The
use of different sets of reagents did not alter the interpreted
neutralization titers. Nine serial dilutions over a range of ~1 to
108 TCID50 were usually enough for performing
the titration of a wide range of neutralizing antibodies.
Analysis of 10 PHS and 50 sera from infants indicated a good
correlation between the neutralization titers obtained by the CCC-ELISA
and those obtained by the NT. The estimates of seropositive samples for
serotypes 1 and 2 were the same. Some sera with predictive negative
signals for serotype 3 in the NT gave positive signals in the
CCC-ELISA. Three of the PHS and four of the sera from infants were
negative by the NT. Some of these negative sera gave positive signals
in the serotype 3 CCC-ELISA. Because of the extensive heterotypic
cross-reactivity among the three types of poliovirus, the effect of the
cross-reactive antibodies on the CCC-ELISA neutralization titers needs
to be further investigated with type-specific antipoliovirus antisera.
Also, the interference of antibodies to other enteroviruses has to be investigated.
In principle, the CCC-ELISA can be used to measure neutralizing
antibodies to a wide range of viruses and is particularly valuable when
the viruses do not produce visible CPE. Furthermore, since the proposed
assay allowed for the detection of the antibody-neutralized viruses in
their equilibrated mixtures, it could be applied to the determination
of the avidity of neutralizing monoclonal antibodies when they are used
instead of polyvalent antisera.
In conclusion, the newly developed CCC-ELISA is nonsubjective, highly
sensitive, and reproducible when used for the detection of neutralizing
antibodies to poliovirus. The CCC-ELISA uses NT reagents, an ELISA
reader, and a commercially available conjugate. In addition, analysis
of the ELISA readings could be automated using software. However, for
evaluation of the CCC-ELISA as an alternative for the standard NT in
epidemiologic studies, an investigation on a large number of sera from
vaccinated humans is essential.
| |
FOOTNOTES |
*
Mailing address: Department of Molecular Biology
National Research Center, Tahrir Street, Dokki, Cairo, Egypt. E-mail:
afwahby{at}hotmail.com. Fax: 202-7603735. Phone: 202-3669980.
| |
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Clinical and Diagnostic Laboratory Immunology, November 2000, p. 915-919, Vol. 7, No. 6
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Top
Abstract
Introduction
