Development of a tissue-culture-based enzyme-immunoassay method for the quantitation of anti-vaccinia-neutralizing antibodies in human sera

Journal of Virological Methods 130 (2005) 15–21

Development of a tissue-culture-based enzyme-immunoassay method for the quantitation of anti-vaccinia-neutralizing antibodies in human sera Osnat Eyal a , Udy Olshevsky a , Shlomo Lustig a , Nir Paran a , Menachem Halevy a , Paula Schneider a , Gil Zomber b , Pinhas Fuchs a,∗ a

Department of Infectious Diseases, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona, Israel b Department of Biotechnology, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona, Israel Received 17 February 2005; received in revised form 26 May 2005; accepted 27 May 2005 Available online 15 July 2005

Abstract Vaccination with vaccinia virus is carried out in order to induce protection against variola virus, the causative agent of smallpox. Serum titer of vaccinia virus-neutralizing antibodies is considered to be well-correlated with in vivo protection. Plaque reduction neutralization test (PRNT) is the gold standard for detecting and quantifying vaccinia virus-neutralizing antibodies in sera of vaccinees. However, PRNT is time and labor consuming, which does not allow large-scale screening needed for a population survey. A simplified, sensitive, standardized, reproducible and rapid method, neutralization tissue-culture enzyme immunoassay (NTC–EIA) was developed for quantitation of neutralizing antibodies against vaccinia virus. The assay consists of the following steps: neutralization of the virus with serially diluted sera, infection of cells in culture and measurement of residual virus replication using an enzyme immunoassay. The assay can be used for animal (rabbit) or human sera. Titer averages obtained using NTC–EIA were highly correlated (R2 = 0.9994) to those obtained using PRNT. The assay is carried out in 96-well plates and takes only 2 days to complete. With the appropriate setup, it can be automated fully to allow screening of a large number of sera. © 2005 Elsevier B.V. All rights reserved.

1. Introduction The determination of neutralizing antibodies levels in the sera of vaccinees allows follow-up of immunization efficacy and protection levels, thus enabling planning of vaccination regimes. The “gold standard” method used for determining antiviral neutralization titer is the plaque reduction neutralization test (PRNT), based on plaque assay. PRNT is used for the determination of neutralization titers for anti-vaccinia virusantibodies because it is specific, direct and reproducible (Orr et al., 2004). In addition, the method is sensitive since it can detect changes caused by neutralization of as little as 100 plaque forming units (pfu) or less (Morens et al., 1985) and the measured unit is as small as 1 pfu. The viral dose and ∗

Corresponding author. Tel.: +972 8 9381607; fax: +972 8 9381639. E-mail address: [email protected] (P. Fuchs).

0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.05.027

the amount of antibodies required for its neutralization are correlated directly. As a result, it is also possible to measure titers of low-neutralizing sera. PRNT, however, has several disadvantages; it is both time and labor consuming and the determination of the neutralized virus is done by counting plaques, rendering the method subjective. This method, therefore, is not suitable for screening large numbers of sera as required for a large population survey. Two new methods were described recently, both using engineered WR strains. Manischewitz et al. (2003) used a ␤-gal expressing WR strain and Earl et al. (2003) have used a GFP expressing WR strain for the quantitation of anti-vaccinia-neutralizing antibodies. An alternative method for quantitation of vaccinianeutralizing antibodies in human sera was, therefore, developed. The requirements from such a method are to maintain the advantages of PRNT and to overcome its shortcomings. It has to be rapid, use relatively small amounts of reagents

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O. Eyal et al. / Journal of Virological Methods 130 (2005) 15–21

and test components and be suitable for automation, which would allow the survey of large populations. Throughout the years, several studies used a tissue-culture immunoassay for determination of neutralizing antibodies raised against a variety of viruses. When used for the determination of anti-rabies-neutralizing antibodies, the method was reported to be rapid, sensitive, easy to perform and reproducible and the results obtained were comparable with those obtained with the standard neutralizing assays in mice. This method allowed screening of a large number of animal sera for the presence of anti-rabies antibodies (Fuchs et al., 1998; Katz et al., 1998). Other tissue-culture-based immunoassays include quantitation of neutralizing antibodies against measles virus (Lee and Taguchi, 1989), poliovirus (Wahby, 2000), Sindbis virus (Lustig et al., 1980) and Japanese encephalitis virus (Ting et al., 2001). These studies too describe the method as very sensitive, reproducible and suitable for viruses that do not induce a cytopathic effect (CPE). Finally, tissue-culture immunoassay for neutralization of antibodies has other advantages over PRNT because it is objective, timesaving and is suitable for automation (Wahby, 2000). In order to optimize viral detection in the assay, various parameters were examined using the WR strain of the vaccinia virus and anti-vaccinia virus anti-serum raised in rabbits. When a system that produced results comparable to those obtained with PRNT was established, the assay was modified for the Lister strain (the vaccine strain used in Israel) and human anti-Lister sera. The assay, termed neutralization tissue-culture enzyme immunoassay (NTC–EIA), is based on infection of cells in 96-well plates with virus that was pre-incubated with anti-serum dilutions and determination of the progeny virus grown in the culture by means of an enzyme immunoassay. NTC–EIA is a rapid, reproducible, sensitive and specific method for the determination of anti-vaccinia virus-neutralizing antibodies in human and animal sera. It is possible to fully automate this method in order to simplify further the test and thus allow surveys of large populations.

2. Materials and methods 2.1. Cells, virus and media Cells: HeLa cells (ATCC CCL 2) were used for virus propagation. Vero (ATCC CCL 8) cells were used for virus stock titration and PRNT. HeLa cells, Vero cells, BSC-1 (ATCC CCL 26) and MRC5 (ATCC CCL 171) cells were tested for NTC–EIA. Cells were maintained according to ATCC recommendations. Viruses: Vaccinia WR (ATCC VR-119), vaccinia Lister (Israeli Health Ministry).

Media: For NTC–EIA Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal calf serum (FCS), 1% l-glutamine, 0.5% combined antibiotics and 1% non-essential amino acids was used (all media and media supplements were purchased from Biological Industries, Beit Ha’Emek, Israel). For PRNT, MEM supplemented with 2% FCS was used for growth and dilution. Overlay medium consisted of MEM containing 2% FCS and 0.4% tragacanth (Sigma, G-1128). 2.2. Anti-sera and conjugates For experiments with the WR strain, the serum of rabbits vaccinated with the virus and control normal rabbit serum were used. Anti-vaccinia virus antibodies were raised in rabbits (R␣VV). Rabbit anti-vaccinia virus–biotin conjugate (R␣VV–B) was a gift from Dr. Y. Gozes. Alkaline phosphatase conjugated with goat anti-rabbit IgG (G␣RIgR–AP) was purchased from Sigma (A-8025). Alkaline phosphatase conjugated with ExtrAvidin (AV–AP) was purchased from Sigma (E-2636). Human sera were obtained from the Army Health Branch of the Israeli Defense Force and grouped into three pools based on titers previously determined by PRNT. These pools were designated: H—sera exhibiting high neutralization titers; L—sera exhibiting low neutralization titers; M—sera exhibiting intermediate neutralization titers. Each pool was composed of five different sera obtained from volunteers revaccinated with the Lister strain of vaccinia virus (Orr et al., 2004). All sera were heat-inactivated at 56 ◦ C for 30 min prior to pooling. Two control human sera were also included, a standard serum containing neutralizing antibodies to vaccinia virus with a PRNT titer of 110 (positive) and a second serum of a non-vaccinated individual (negative). 2.3. Plaque reduction neutralization assay Sera were diluted serially to six 2-fold dilutions at the range from 1:10 to 1:5120, depending on the pool’s expected titer. Tubes containing 350 ␮l of each dilution and an equal volume of Lister virus (103 pfu/ml) were mixed. Following incubation at 37 ◦ C, 5% CO2 for 1 h, 200 ␮l was added to each well of Vero cells monolayer grown overnight in 12-well plates. Virus was adsorbed for 1 h under the same conditions, after which 2 ml of overlay medium was added and the plates were further incubated at 37 ◦ C, 5% CO2 for 3 days. The medium was then aspirated; the monolayer was fixed with 70% ethanol, stained with 0.3% basic fuchsin (Sigma, P-1528, in 95% ethanol 5% phenol solution) and the number of plaques in each well was determined. Each dilution was tested in triplicate. The following controls were included in each assay: non-neutralized virus, negative serum at dilution 1:10 and positive serum at dilutions from 1:20 to 1:320. The 50% reduction point (NT50 )

O. Eyal et al. / Journal of Virological Methods 130 (2005) 15–21

was calculated using the Spearman–Karber formula (Finney, 1952). 2.4. NTC–EIA During the development stages of the assay, the following parameters were tested: strain of vaccinia virus and cell line, incubation time, fixation method, blocking and dilution solutions. Two vaccinia virus strains were tested: WR and Lister. Cell lines tested were: Vero, MRC-5 and BSC-1. All cells were added as a trypsinized suspension. Viral progeny were allowed to grow in the cells for 24 or 48 h post-infection. Fixation methods tested were: 80% acetone in water, 1:1 acetone:methanol and 3.7% formaldehyde followed by 80% acetone. Blocking and dilution solutions tested were: 1% bovine serum albumin (BSA) fraction V (Sigma A-8022), 2.5% skim milk, 5% Similac, 0.5% Similac, Vero cell lysate consisting of 20% FCS and 40% supernatant of lysed Vero cells. All blocking solutions were prepared in PBST (PBS–0.05% Tween 20). Eight 2-fold dilutions of each serum at the range from 1:5 to 1:2560, depending on the expected pool’s titer were prepared at a final volume of 250 ␮l. The 50 ␮l of diluted serum was distributed into each of four corresponding wells in a 96-well plate. An equal volume of vaccinia virus was added to each well and the plates were incubated at 37 ◦ C, 5% CO2 for 1 h. Following incubation, 50 ␮l of cells was added to each well. The plates were placed on a plate shaker for 1 min and incubated at 37 ◦ C, 5% CO2 . After 24–48 h incubation, supernatant was aspirated and cells were fixed at room temperature for 30 min. Wells were then blocked at 37 ◦ C for 1 h. The blocking solution was then aspirated and 50 ␮l of R␣VV-B diluted 1:1000 was added to each well. Plates were incubated again at 37 ◦ C for 1 h. Following incubation, the plates were washed five times with PBST and 50 ␮l of AV–AP diluted 1:1250 was added to each well. Plates were incubated at room temperature for 20 min and washed again five times with PBST. The 50 ␮l of p-nitrophenyl substrate (Sigma, N-1891) were then added to each well and plates were incubated at room temperature for 30 min, after which absorbance was determined at 405 nm using a SpectraMAX 190 ELISA reader (Molecular Devices Corporation, Sunnyvale, CA, USA). Every assay included a negative control (serum from a non-vaccinated individual) and each plate contained a virus calibration curve including a virus standard (100%) consisting of the same viral infecting dose used in the neutralization wells. The virus calibration curve was used to ascertain regularity of the test and the presence of a linear dynamic range. NT50 was determined using the Spearman–Karber formula (Finney, 1952). In all experiments, the detection limit was considered to be an absorbance value greater than twice the background (non-infected cells).

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2.5. Statistical analysis The neutralization titers of sera tested obtained by both PRNT and NTC–EIA were compared. Variance between averages in both methods was calculated using Student’s t-test and variance of experiments within each group was calculated using the F-test. 3. Results The NTC–EIA is composed of two parts: the neutralization of the virus and the detection of non-neutralized virus progeny present in cells following incubation. The results describe the development of the assay. When the optimal conditions for viral progeny detection were established, the assay was tested for viral neutralization titers of animal and human sera. The results were compared to those obtained using PRNT. 3.1. Determination of viral strain and incubation time The Lister strain is used for vaccine production in Israel, but since human sera were scarce and the initial experiments designed to establish the parameters for the TC–EIA were carried out using the WR strain, rabbit sera raised against the WR strain of vaccinia virus were used. Using the WR strain, results were obtained after only 24 h. However, since the progression of infection with the Lister strain is slower, in later experiments, when the initial parameters for the assay were established, post-infection incubation time with the Lister strain was changed to 48 h. 3.2. Determination of cell line Several cell lines were compared for use in NTC–EIA (Table 1A–C). Detection of viral antigen in Vero cells was carried using R␣VV antibodies followed by incubation with G␣RIgG–AP conjugate (Table 1A). The use of this cell line was discontinued due to the high background obtained. The detection limit was high, 103 pfu or more and there was inconsistency among tests (not shown). Two other cell lines, BSC-1 and MRC-5 were tested using a different detection system, consisting of biotinylated rabbit anti-vaccinia virus followed by avidin conjugated to AP (Table 1B and C). It was found that MRC-5 cells were not suitable for this assay due to the high background. Infection of these cells also resulted in a narrow dynamic range and poor reproducibility both within different wells in a single test and among different tests (Table 1B). BSC-1 cell line was chosen for further experiments because although the background was not low, the reproducibility was better than in MRC-5 cells, which allowed the detection of less than 100 pfu (Table 1C). Since BSC-1 cells have slow growth rate, 23,000 cells/well in a 96-well plate are needed to produce a monolayer that allows detection of viral proliferation following a 2-day incubation. Shorter incubation periods were examined, but 24 h incubation was not suffi-

O. Eyal et al. / Journal of Virological Methods 130 (2005) 15–21

18 Table 1 Effect of cell type on assay sensitivity (A) Vero cells pfu/well

Absorbance at OD405

104

0.771 0.557 0.263 0.178

103 102 10

± ± ± ±

0.031 0.032 0.021 0.008

Control

Remarks (Vero cells)

0.190

High background Good reproducibility Detection of ≥103 pfu

(B) MRC-5 cells pfu/well

103 102 10

Plate 1

Plate 2

Absorbance at OD405

Control

Absorbance at OD405

Control

Remarks (MRC-5 cells)

0.368 ± 0.058 0.218 ± 0.005 0.136 ± 0.008

0.175

0.173 ± 0.061 0 ± 0.017 0 ± 0.019

0.006

High background Poor reproducibility Detection of 103 pfu

(C) BSC-1 cells pfu/well

103 102 10

Plate 1

Plate 2

Absorbance at OD405

Control

Absorbance at OD405

Control

Remarks (BSC-1 cells)

0.446 ± 0.034 0.187 ± 0.023 0.103 ± 0.006

0.082

0.471 ± 0.071 0.254 ± 0.017 0.113 ± 0.009

0.109

High background Good reproducibility Detection of <102 pfu

Effect of cell line on the detection limit of vaccinia virus. Vero cells (A), MRC-5 cells (B) or BSC-1 cell (C) were seeded in 96-well plates and inoculated with vaccinia virus WR strain at varying viral concentrations. Following 24 h incubation at 37 ◦ C, 5% CO2 , the cells were fixed with 80% acetone and the amount of viral antigen was determined by EIA as described in Section 2. Detection of virus in Vero cells experiment was performed using the rabbit-anti-vaccinia virus + goat-anti-rabbit IgG–AP system. MRC-5 and BSC-1cells were compared with the R␣VV–biotin + AV–AP system. For MRC-5 and BSC-1 cells, results of two plates for each cell line are depicted to demonstrate consistency between plates.

cient to distinguish the infected cells from the background and comparison of 30 and 48 h incubation favored the longer incubation period due to broader dynamic range. 3.3. Determination of viral infecting dose In order to determine the optimal viral infecting dose for the test, various amounts of virus were tested. Fig. 1 depicts the direct correlation between viral input and absorbance at

405 nm for the Lister strain. The 75 pfu (MOI = 0.0033) was chosen as the optimal virus infecting dose, as it is within the dynamic range of viral infecting dose (10–100 pfu/well) – a range, where the increase in absorbance is linearly correlated with the increase in viral infecting dose. This dose is, on one hand, large enough to produce a high signal and thus permits the evaluation of reduction in viral progeny following neutralization and on the other hand, it is low enough to render the assay sensitive. 3.4. Determination of fixation method and of blocking and dilution solutions Three fixation methods were examined: 80% acetone in water, 1:1 methanol:acetone and 3.7% formaldehyde + 80% acetone. The three methods were compared for reduction of background, increase in dynamic range and in reproducibility. Fixation with 80% acetone was chosen for the assay, because it best preserved viral antigenicity and had a broader dynamic range (results not shown). Of the various solutions examined, 1% BSA in PBST gave the optimal results as a diluent and as a blocking reagent (results not shown).

Fig. 1. Effect of infectious dose of vaccinia virus on viral antigen expression in BSC-1 cells as measured by TC–EIA. The 23,000 BSC-1 cells/well in a 96-well plate were infected with varying amounts of the Lister strain of vaccinia virus and incubated for 48 h followed by fixation with 80% acetone and EIA as described in Section 2.

3.5. Determination of neutralization titers of human sera pools by PRNT Neutralization titers of the three pools of human sera from re-vaccinated volunteers (H, M and L) were determined in

O. Eyal et al. / Journal of Virological Methods 130 (2005) 15–21 Table 2 NT50 values for three human sera pools obtained in two methods

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Table 3 Statistical comparison of NTC–EIA and PRNT using t- and F-test

Experiment

Pool H

Pool M

Pool L

PRNT #1 PRNT #2 PRNT #3 PRNT #4 PRNT #5

942 1541 1457 1011 1376

431 616 475 478 581

49 40 40 30 38

PRNT average

1265 ± 271

516 ± 78

39 ± 7

NTC–EIA #1 NTC–EIA #2 NTC–EIA #3

1617 845 1075

453 366 488

35 27 36

NTC–EIA average

1179 ± 396

436 ± 63

33 ± 5

NT50 titers of three human sera pools calculated in PRNT and NTC–EIA as described in Section 2.

five separate experiments as 1265 for pool H, 516 for pool M and 39 for pool L (Table 2). 3.6. Determination of neutralization titers of human sera pools by NTC–EIA The neutralization titers of the three pools were determined in three separate experiments as 1179 for pool H, 436 for pool M and 32 for pool L (Table 2). The limit of detection as shown by the negative control serum was calculated as 1:20 (data not shown). Fig. 2 depicts the results of an experiment in which neutralization of the Lister strain of vaccinia virus by human sera (pools H and L) was measured by NTC–EIA. The neutralization efficiency of pool L is much smaller than that of pool H as viral antigens reach levels similar to those of non-neutralized virus (100%) at a much lower serum dilution. Fig. 2 also depicts the graphic determination of the neutralization titer, which correlates well with the titer calculated by the Spearman–Karber method (Table 2).

Fig. 2. Neutralization of human sera using NTC–EIA. The 23,000 BSC1 cells/well in a 96-well plate were infected with 75 pfu of vaccinia virus Lister strain neutralized with diluted human sera (pools H and L). Neutralization and determination of viral replication were performed as described in Section 2. Dotted lines depict 100 and 50% value of wells without serum (virus control). Dotted arrow depicts a graphic calculation of NT50 titer.

Pool

L M H





P-value

T-value

P-value

F-value

0.166 0.171 0.759

1.62 1.60 0.34

0.751 0.854 0.467

1.882 1.557 0.468

Comparison of titers’ averages obtained in both NTC–EIA and PRNT using t-test. Averages are statistically similar (P > 0.05). Comparison of titer variances obtained in both NTC–EIA and PRNT using F-test. Variance within each group is statistically similar (P > 0.05).

3.7. Comparison of PRNT and NTC–EIA Fig. 3 depicts the correlation between the average NT50 values obtained by both methods. Each dot represents a data pair in which values on the X-axis were measured by PRNT and on the Y-axis, values were determined in NTC–EIA. The linear correlation curve has a high correlation coefficient (R2 ) of 0.9994. 3.8. Statistical comparison between PRNT and NTC–EIA Statistical comparison between the two neutralization methods was carried out in order to determine the validity of NTC–EIA. Comparison of titers in t-test, depicted in Table 3, revealed that average titers in both methods were similar (P-value > 0.05). The variance in test results was calculated using F-test. The variance within each group was found to be similar for all pools (P-value > 0.05) for the two neutralization methods (Table 3). Results of both F- and t-test indicate that under the experimental conditions developed, a high correlation exists between the two methods.

Fig. 3. Correlation between neutralization titers obtained in PRNT and NTC–EIA. Correlation between neutralization titers (NT50 ) of the three pools of human sera obtained in both methods. Each dot represents the average of at least three experiments.

O. Eyal et al. / Journal of Virological Methods 130 (2005) 15–21

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Table 4 Description of the final format of NTC–EIA neutralization assay for human sera Step Viral neutralization, infection of cells and replication Dilution of sera

Description Preparation of eight 2-fold dilutions in test tubes in a final volume of 250 ␮l. Preparation of 250 ␮l medium for non-neutralized virus. A validated control serum with established NT titer should be included in each assay plate as a standard

Administration of sera into plates

Dispensing of 50 ␮l of each diluted serum into a corresponding series of four wells in a flat-bottom 96-well plate

Administration of virus and neutralization

Dispensing of 50 ␮l containing 75 pfu of Lister strain of vaccinia virus to each well. Incubation at 37 ◦ C, 5% CO2 for 1 h

Addition of cells and viral replication

Addition of 50 ␮l containing 23,000 BSC-1 cells to each well. Incubation at 37 ◦ C, 5% CO2 for 48 h

Immunoassay Fixation

Aspiration of supernatant and fixation with 100 ␮l/well of 80% cold (−20 ◦ C) acetone. Incubation at room temperature (20 ◦ C) for 30 min

Blocking

Single wash with PBS and addition of 250 ␮l/well PBST + 1% BSA. Incubation for 1 h at 37 ◦ C

Addition of biotinylated antibodies

Aspiration of supernatant. Addition of 50 ␮l/well of rabbit-raised biotin-conjugated anti-vaccinia virus-antibodies diluted 1:1000 in PBST + 1% BSA to each well. Incubation at 37 ◦ C for 1 h

Addition of alkaline phosphatase conjugate

Five washes with PBST. Addition of 50 ␮l/well of avidin–alkaline phosphatase conjugate diluted 1:1250 in PBST + 1% BSA at room temperature for 20 min

Addition of substrate

Five washes with PBST. Addition of 50 ␮l of pNPP substrate to each well. Incubation at room temperature for 30 min

Determination of absorbance

Determining absorbance at 405 nm

4. Discussion Vaccination with vaccinia virus is used for protection against smallpox. Following a vaccination campaign launched by the Israeli health authorities in 2002–2003, in which PRNT and ELISA were used for assessment of vaccination success (Orr et al., 2004), the need for a simplified assay allowing survey of a large population arose and it was decided to develop an ELISA-based tissue-culture neutralization assay which can be automated later. Micro-neutralization assays on cells have been reported previously for various viruses (Fuchs et al., 1998; Katz et al., 1998; Lee and Taguchi, 1989; Lustig et al., 1980; Ting et al., 2001; Wahby, 2000). The development of a new neutralization assay for replacement of a ‘gold standard’ method needs to address several parameters, such as detection limits, reproducibility, specificity and correlation with the PRNT gold standard method. Several variables affecting these parameters: cell line, viral infecting dose, incubation time, fixation method and blocking and dilution reagents were tested and the optimal conditions were determined. Cells were chosen based on their ability to allow viral replication that would permit detection of comparatively small viral inocula following a 24–48 h incubation with a dynamic range of 10–150 pfu. Three cell lines, Vero, MRC-5 and BSC-1 were tested for compatibility with NTC–EIA. Of the three, BSC-1 cells allowed the detection of viral replication after 48 h, had a low detection limit (10 pfu)

and the results obtained with these cells were reproducible (Table 1C). The determination of detection limit and viral infecting dose is important because the infecting dose has to be a small one in order to increase the assay’s sensitivity. However, the infecting dose should be high enough to produce a signal that will allow monitoring of reduction due to neutralization. In this assay, the dynamic range, in which an increase in virus inoculum caused an increase in absorbance, was found to be between 10 and 150 pfu. The number of pfu chosen for neutralization was 75 pfu (MOI = 0.0033), which is within the dynamic range (Fig. 1) and gave results similar to those obtained by PRNT. Fixation of cells prior to EIA is necessary to inactivate viable virus, expose viral antigens and prevent cell monolayer from detaching during washes, while preserving antigenicity. Among the three methods tested, only acetone allowed lower background and distinction between various MOIs within the dynamic range. In order to test the NTC–EIA method, human sera were pooled according to their PRNT titers into three pools: high, intermediate and low. Comparison of the pools’ neutralization titers obtained by NTC–EIA to those obtained by PRNT revealed a high correlation (Fig. 3). Statistical calculations and comparison of the two methods strengthened the validity of the NTC–EIA due to similarities in average values, variance within each method and between the two methods (Table 3).

O. Eyal et al. / Journal of Virological Methods 130 (2005) 15–21

The final suggested format of the NTC–EIA assay is described in Table 4. This format can be easily changed to accommodate any specific needs, i.e. number of serum dilutions, adaptation to screening of anti-viral drugs, etc. In order to achieve high sensitivity in neutralization tests, it is important that the virus strain used in the test will be the same virus used for vaccination. Our assay has the flexibility to accommodate any vaccinia strain and is simpler than other recently suggested methods (Manischewitz et al., 2003; Earl et al., 2003) because it is not based on genetically modified virus and requires only standard laboratory equipment. In conclusion, a simple, objective, reproducible, time and labor saving tissue-culture-based neutralization assay was developed for the determination of anti-vaccinia virus titers of neutralizing antibodies in human and animal sera. Results obtained with NTC–EIA correlate strongly with titer averages obtained in the ‘gold standard’ PRNT method (R2 = 0.9994). Similar results were obtained for rabbit serum, where NT50 of anti-serum raised against WR strain was determined as 1:3136 by NTC–EIA and 1:4422 by PRNT (results not shown). The assay (as TC–EIA) could also be used for virus titration and is very sensitive, as its detection limit is 10 pfu. NTC–EIA could be used to assess neutralizing antibodies in sera of vaccines with the lowest detection limit of 1:20 and for the evaluation of anti-viral compounds in various samples. Another advantage of NTC–EIA and TC–EIA is that it could be used to assess antibodies raised against viruses that do not produce CPE and for the titration of these viruses. Since it is carried out in 96-well plates, several sera could be tested on a single plate and results read simultaneously. With the appropriate setup, this assay could be fully automated. All these factors render on NTC–EIA suitable for large-scale screening.

Acknowledgements We thank Dr. N. Orr and Dr. D. Cohen from the Army Health Branch of the Israeli Defense Force and Dr. H. Marcus

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from the Israel Institute for Biological Research for supplying human sera.

References Earl, P.L., Americo, J.L., Moss, B., 2003. Development and use of a vaccinia virus neutralization assay based on flow cytometric detection of green fluorescent protein. J. Virol. 77, 10684–10688. Finney, D., 1952. Statistical Methods in Biological Assays. Charles Griffin & Company Limited, London. Fuchs, P., Zamir, S., King, R., Freeman, E., Davidson, M., Aubert, M., Katz, D., 1998. Detection of anti-rabies neutralizing antibodies by tissue culture–enzyme immunoassay (NTCEIA) in rodents and canines after oral vaccination with a vaccinia-rabies recombinant vaccine. Isr. J. Vet. Med. 53, 143–153. Katz, D., Freeman, E., Davidson, M., Abramson, M., Fuchs, P., 1998. Detection of anti-rabies neutralizing antibodies in humans and cattle by a combined tissue culture and enzyme linked immunoassay. Isr. J. Vet. Med. 53, 132–142. Lee, S.M., Taguchi, F., 1989. Rapid identification and typing of herpes simplex virus by a new enzyme immunoassay with peroxidase-labeled complement C1q. Arch. Virol. 108, 247–257. Lustig, S., Shapira, A., Katz, D., 1980. Neutralization of Sindbis virus in tissue culture by radioimmunoassay. Isr. J. Med. Sci. 16, 473. Manischewitz, J., King, L.R., Bleckwenn, N.A., Shiloach, J., Taffs, R., Merchlinsky, M., Eller, N., Mikolajczyk, M.G., Clanton, D.J., Monath, T., Weltzin, R.A., Scott, D.E., Golding, H., 2003. Development of a novel vaccina-neutralization assay based on reporter-gene expression. J. Infect. Dis. 188, 440–448. Morens, D.M., Halstead, S.B., Repik, P.M., Putvatana, R., Raybourne, N., 1985. Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization. J. Clin. Microbiol. 22, 250–254. Orr, N., Forman, M., Marcus, H., Lustig, S., Paran, N., Grotto, I., Klement, E., Yehezkelli, Y., Robin, G., Reuveny, S., Shafferman, A., Cohen, D., Study Group Medical Corps Israel Defense Force, Study Group Israel Institute for Biological Research, 2004. Clinical and immune responses after revaccination of Israeli adults with the Lister strain of vaccinia virus. J. Infect. Dis. 190, 1295–1302. Ting, S.H., See, E., Tan, H.C., Lee, M.A., Ooi, E.E., 2001. Development of a simplified assay for the detection of neutralizing antibodies to Japanese encephalitis virus. J. Virol. Methods 93, 43–47. Wahby, A.F., 2000. Combined cell culture enzyme-linked immunosorbent assay for quantification of poliovirus neutralization-relevant antibodies. Clin. Diagn. Lab. Immunol. 7, 915–919.