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Rapid in vitro micro-cytotoxicity tests for the detection and quantitation of neutralizing antibodies to both viruses and toxins
Journal oflmmunological Methods, 166 (1993) 157-164
157
© 1993 ElsevierSciencePublishers B.V. All rights reserved0022-1759/93/$06.00
JIM06850
Rapid in vitro micro-cytotoxicity tests for the detection and quantitation of neutralizing antibodies to both viruses and toxins C. van de W a t e r a, E.A. van D u r a a, J.G.M.M. van der Stap a, R. Brands b and W.J.A. B o e r s m a .,a Department of Immunology and Medical Microbiology, T.N.O. Medical Biological Laboratory, P.O. Box 5815, 2280 HV Rijswijk, Netherlands, and b Solvay / Duphar, P.O. Box 900, 1380 DA Weesp, Netherlands (Received 8 June 1993, revised received 5 July 1993, accepted 6 July 1993)
A generally applicable method was developed for the rapid and quantitative detection of both toxinand virus neutralizing antibodies. The method was optimized for three different biological agents, i.e., Shigella toxin, influenza viruses (A/Beying, A / T a i w a n and B/Yamagata) and Chikungunya virus. The in vitro micro-cytotoxicity tests developed for the detection and quantitation of neutralizing antibodies are based on the inhibition of the virus- or toxin-induced cytotoxic effect by antibodies. As a result of the cytotoxicity, infected cells are no longer attached to the solid phase and can be easily removed. Thereafter, the proteins of the remaining living cells are stained. After removing the excess dye, the remaining dye is dissolved and the absorbance values are measured. The neutralization titers are determined from the absorbance values. Since the tests are performed in wells of microtiter plates, the in vitro micro-cytotoxicity tests are less laborious and consume less reagent in comparison with classical neutralization tests. Key words: Neutralizing antibody; Detection; Cytotoxicity; Protection; Immune response; Vaccine efficacy; Shigella toxin; Influenza virus; Chikungunya virus
Introduction
In order to estimate the efficacy of vaccines in protection against pathogens or toxins, it is usual to determine the ability of the evoked serum antibodies to neutralize cytopathogenic effects.
* Corresponding author. Tel.: +31 15 843188; Fax: +31 15 843989. Abbreviations: BCA, bicinchoninic)acid; BHK, baby hamster kidney; DMEM, Dulbecco's modification of Eagle's medium; ELISA, enzyme-linked immunosorbent assay; FRN, focus reduction neutralization; HA, hemagglutinin; HAl, hemagglutination inhibition; MDCK, Madin-Darby canine kidney; N, neutralization; pfu, plaque forming unit(s); PBS, phosphatebuffered saline; SRD, single radial diffusion.
Furthermore, detection and quantitation of the neutralizing antibody response may also be a valuable tool for the determination of the humoral immune status of humans or animals after exposure to biological agents. Consequently, the level of circulating antibodies with neutralizing capacity indicates the immune responsiveness of individuals a n d / o r the immunogenicity of the antigen. Classical methods, such as the conventional plaque reduction assays for antiviral antibodies, can be used for quantitation of the neutralizing antibody response but these methods are both expensive and time-consuming and the results are obtained by visual interpretation. The focus reduction neutralization (FRN) assays (Bernstein et al., 1989; Okuno et al., 1990) are
158
performed in 96-well tissue culture plates and are based on the detection and enumeration of foci stained by means of enzyme immunochemical techniques. These tests are less costly, less laborious and generally more reproducible. The results, however, are obtained after visual observation. Modified FRN assays, i.e., the micro-neutralization immunoassays (Knowlton et al., 1991; Tomiyama et al., 1993) combine the advantages of both FRN assays and ELISA. In micro-neutralization immunoassays the results are easily quantifiable because they are obtained by means of an ELISA reader instead of visual estimation. Therefore, the micro-neutralization immunoassay represents an improvement over the FRN-assay. However, both the FRN-assay and the microneutralization immunoassay require specific antibody preparations and are limited to the detection of neutralizing antiviral antibodies. In this paper in vitro micro-cytotoxicity tests for screening of neutralizing antibodies are described. These tests are based on the cytotoxicity assay as described for the quantitation of Shigella toxin (Gentry and Dalrymple, 1980). In comparison with the micro-neutralization immunoassay, the present in vitro micro-cytotoxicity tests have the advantage of (i) broad applicability, and (ii) ease of optimization for other biological agents. This means that the in vitro micro-cytotoxicity tests can be used for screening neutralizing antibodies to both viruses and toxins. Since specific antibody preparations are not required for the detection and quantitation of neutralizing antibodies, the in vitro micro-cytotoxicity tests can easily be broadened to other biological agents which have a cytotoxic effect on target cells growing on adherent monolayers. This is demonstrated in this paper by application of the in vitro micro-cytotoxicity tests for screening neutralizing antibodies to three totally different biological agents, i.e., Shigella toxin, influenza viruses and Chikungunya virus. Materials and methods
Vaccines, uiruses, toxin and cells Trivalent embryonated egg derived influenza vaccine (1992: A/Beying, A/Taiwan, B/Yamagata) and influenza viruses A/Beying, A / T a i w a n
and B/Yamagata were a kind gift of R. Brands and G. van Scharrenburg (Solvay/Duphar, Weesp, Netherlands). Chikungunya virus was kindly supplied by Y. Okuno (Research Institute for Microbial Diseases, Osaka University, Japan). Shigella toxin was produced as described by Keusch et al. (1988). The 60R strain of Shigella dysenteriae, being used for the production of Shigella toxin was a gift of A.D. O'Brien (Department of Microbiology, Uniformed Services of the Health Sciences, Bethesda, MD, USA). The baby hamster kidney cell line (BHK21 clone 13) was obtained from the American Type Culture Collection. The Madin-Darby canine kidney (MDCK) cell line was obtained from Solvay/ Duphar. The HeLa $3 cell line used was in stock in our laboratory.
Cell culture The culture conditions for target cells being used in the in vitro micro-cytotoxicity tests for measuring anti-Shigella toxin and anti-Chikunkunya neutralizing antibodies, i.e., HeLa ceils and BHK cells respectively, have been described elsewhere (Gentry and Dalrymple, 1980; Davis et al., 1971). Target cells used in the in vitro microcytotoxicity test for measuring anti-influenza virus neutralizing antibodies, i.e., MDCK cells, have been described by Wood et al. (1989). Cell cultures of BHK, MDCK and HeLa cells were maintained in 25 cm 2 culture flasks at 37°C in a 5% CO 2 atmosphere in complete RPMI, serum-free EpiSerf medium and Dulbecco's modification of Eagle's medium (DMEM) with Ham's F-10 respectively. Complete RPMI medium consisted of RPMI 1640 medium with 106 IU penicillin/ml, 10 mg streptomycin/ml, 5 x 10 -5 M/3-mercaptoethanol and 10% fetal calf serum; serum-free EpiSerf medium was supplemented with 1.5 mg NaHCO3/ml, 500 IU penicillin/ml, 500 IU streptomycin/ ml and 0.5 mg glutamine/ml; D M E M / F 10 medium, 95 : 100 (v/v) was supplemented with 10% fetal calf serum, 110 IU penicillin/ml, 110 IU streptomycin/ml, 0.17 mg glutamine/ml and 2.3/zg hypoxanthine/ml. Cells were passaged every 5-7 days by the addition of trypsin-EDTA, passed into new 25 cm 2 culture flasks (2 x 10 4 BHK cells, 5 × 104
159 HeLa cells and 8 × 104 MDCK cells respectively) and incubated at 37°C under 5% CO 2 atmosphere. Sera The sera screened for neutralizing antibodies were obtained from mice and rabbits immunized with toxin- and virus-derived immunogens. Trivalent embryonated egg derived influenza vaccine (Influvac, Solvay/Duphar), inactivated Chikungunya virus and inactivated Shigella toxin were used as immunogens. Chikungunya virus was inactivated by UV irradiation as described by Nakoa and Hotta (1973). A single low pressure mercury vapour lamp (Phillips, 15 W, TUV) with an incidence fluence rate of 0.604 W / m 2 at 254 nm was used for 10 min to obtain complete inactivation of Chikungunya virus. Shigella toxin was inactivated by a 30 h treatment with 0.1% formaldehyde as described by Donohue-Rolfe et al. (1984). Groups of 12-week-old female B A L B / c mice were injected subcutaneously with influenza vaccine (0.94-60/~g), and intraperitoneally with inactivated Shigella toxin (50 /zg) and inactivated Chikungunya virus (50 /zg) respectively. Rabbits were injected intradermally with inactivated Chikungunya virus (200 Izg). Influenza vaccine was used without adjuvant. For the immunization of mice the Chikungunya virus and Shigella toxin preparations were emulsified in 'specol', i.e., a water-in-oil suspension (Bokhout et al., 1981; Boersma et al., 1992); booster immunizations were given 21 days after priming. For the immunization of rabbits the Chikungunya virus preparation was emulsified in complete Freund's adjuvant for priming and in incomplete Freund's adjuvant for booster injections. The booster injections were given 28 days after priming. The amounts of influenza vaccine were based on hemagglutin antigen as determined with the single radial diffusion (SRD) test; the amounts of inactivated Chikungunya virus and inactivated Shigella toxin were based on the results obtained with the BCA protein determination as described by Smith et al. (1985). Monoclonal antibodies Monoclonal antibodies specific for Shigella toxin and Chikungunya virus were produced by
hybridomas obtained from the fusion of the mouse SP2/0 plasmacytoma cell line with spleen ceils from BALB/c mice which were immunized with inactivated Shigella toxin or inactivated Chikungunya virus. The procedure followed was identical to the procedure described by Zegers et al. (1991) for the preparation of monoclonal antibodies to a~-antitrypsin. Plaque test The plaque test for Chikungunya virus was performed in an analogous manner to the procedure described by Matsumura et al. (1972). BHK monolayers were incubated with 100 /zl volumes of Chikungunya virus dilutions at 37°C, 5% CO 2 for 1 h. Overlay medium (RPMI containing 1.6% agarose) was added. After 3 days incubation at 37°C, 5% CO 2 the cultures were stained and fixed with a solution containing 0.2% crystal violet and 0.8% ammonium oxalate. After removing of the overlay medium and washing of the stained ceils with demineralized water, the plaques were counted. Hemagglutination inhibition test Hemagglutination inhibition (HAD tests were performed in microtiter plates by standard methods. After preincubation of serum dilutions with 2 HA units of influenza virus A / T a i w a n (50 /xl) for 1 h at 37°C, 100/~1 volumes of a 0.5% chicken erythrocyte suspension in physiological saline solution were added. The microtiter plate was incubated for 1 h at 4°C and the hemagglutination titer, defined as the reciprocal of the serum dilution at which agglutination just disappeared, was read by eye. To eliminate serum factors which could inhibit non-specific agglutination, the sera were pretreated with receptor-destroying enzyme (Conrath, 1972). In vitro micro-cytotoxicity test MDCK cells with supplemented serum-free EpiSerf medium, BHK cells with complete RPMI medium and HeLa ceils with supplemented DMEM/F-10 medium were used in the in vitro micro-cytotoxicity tests for quantitation of influenza virus, Chikungunya virus and Shigella toxin respectively. The in vitro micro-cytotoxicity tests were based on the cytotoxicity tests previ-
160 ously described for the quantitation of Shigella toxin (Gentry and Dalrymple, 1980). For the detection and quantitation of influenza virus-, Chikungunya virus- and Shigella toxin neutralizing-antibodies, preincubations of virus or toxin with serum/supernatant dilutions were performed. Optimized in vitro micro-cytotoxicity tests for measuring neutralizing antibodies were performed as follows. Serial two-fold dilutions of the sera under investigation were prepared in the wells of a 96-well microtiter plate (Costar, Cambridge, MA, USA) using supplemented EpiSerf medium, complete RPMI and supplemented DMEM/F-10 medium as dilution buffers for the influenza virus, Chikungunya virus and Shigella toxin neutralization assay respectively. Influenza virus A / T a i w a n (50 ~l 40 HA U / m l ) , Chikungunya virus (50 /~1 480 p f u / m l ) and Shigella toxin (50/.d 126 ng/ml) respectively were added to 50 ~l volumes of the serum dilutions. After incubation for 1 h at 37°C, 100/~l volumes of freshly trypsinized cells resuspended in medium (4 × 105 M D C K / m l EpiSerf; 2 × 105 B H K / m l RPMI; 1 × 105 H e L a / m l DMEM/F-10) were added. After incubation at 37°C, 5% CO 2 ('Shigella toxin' 48 h; 'Chikungunya virus' 28 h and 'influenza viruses' 24 h) the medium was aspirated. The wells of the microtiter plates were washed twice with 200 ~l of PBS. The residual living cells were fixed and their proteins stained with 50 ~l of a solution of 0.5% 2.00
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dilution curves were obtained for several Shigella toxin concentrations, i.e., 630 ng/ml (D), 126 ng/ml (+), 25 ng/ml (o) and 5 ng/ml ( Ix).
Amido black in methanol/demineralized water/ acetic acid, glacial (5 : 5 : 1). After 20 min incubation at room temperature the staining solution was removed and the wells were subsequently washed with destaining solution, i.e., methanol/ demineralized water/acetic acid, glacial (5 : 5 : 1) and demineralized water. The protein-associated dye was dissolved in 100 /~I of PBS/ethanol (1:1) and the absorbance values were measured at 580 nm using an ELISA reader (Flow Titertek Multiskan 8 channel reader, Flow Laboratories, Irvine, Scotland). The neutralization titers, defined as the reciprocal of the serum dilutions giving a 50% reduction of the cytotoxic effect, were calculated.
Results
Optimum conditions for in vitro micro-cytotoxicity tests In vitro micro-cytotoxicity tests were developed for both Shigella toxin, influenza viruses (A/Beying, A / T a i w a n and B/Yamagata) and Chikungunya virus. Initially the optimal cell-culture conditions for Shigella toxin, influenza virus and Chikungunya virus sensitive cells, i.e., HeLa, MDCK and BHK cells respectively, were established. The cytotoxicity tests were optimized for cell concentration and incubation time. For Shigella toxin, influenza viruses and Chikungunya virus, the optimized incubation times were established to be 48 h, 24 h and 28 h respectively. In case of Chikungunya virus, the sensitivity of the cytotoxicity test could be easily improved by increasing the incubation time to 48 h and lowering the cell concentration to 0.5 × 105 BHK cells/ml. This phenomenon must be explained by infection of BHK cells with virus particles produced after the first round of replication. However, the sensitivity of the influenza virus cytotoxicity tests could not be further increased by prolonging the incubation time and lowering the cell concentration. To validate the cytotoxicity test for Chikungunya virus, this assay was compared with a classical plaque test. The amount of Chikungunya virus resulting in a 50% cytotoxic effect in the cytotoxicity test corresponded to six plaque forming units (pfu) in the classical plaque test. This
161 suggested that the sensitivities of both tests were similar. The 50% cytotoxic value in the cytotoxicity test indicated that 50% of the cells were infected, i.e., 1 x 104 cells. Extrapolation of these results to the classical plaque test showed that the number of pfu measured (i.e., six) was rather low in comparison with the maximum number of pfu which could be obtained (i.e., 104). The optimal virus and toxin concentrations in the in vitro micro-cytotoxicity tests for the detection and quantitation of neutralizing antibodies, i.e., defined as the virus or toxin concentrations at which 90% of the ceils were detached from the solid-phase, were established by means of doseresponse curves in the cytotoxicity tests.
In vitro micro-cytotoxicity tests The in vitro micro-cytotoxicity tests were very useful for the development of immunochemical methods, in particular with respect to screening and selection of polyclonal and monoclonal antibodies. The in vitro micro-cytotoxicity tests were successfully applied for the selection of neutralizing monoclonal antibodies to both Shigella toxin and Chikungunya virus. A dose-response curve of a Shigella toxin neutralizing monoclonal antibody is presented in Fig. 1. Since non-specific interactions caused by normal mouse serum or monoclonal antibodies to other biological agents were not observed (results not shown), the dose-response dependency presented in Fig. 1 could be completely attributed to the specific neutralizing capacity of the monoclonal antibody. The protective capacity of this monoclonal antibody is indicated by the high neutralization titer (640 for Shigella toxin 126 n g / m l ) . The application of this monoclonal antibody in immunochemical methods for the determination of Shigella toxin will be the subject of further investigation. Influenza virus neutralizing antibodies in sera obtained from mice immunized with trivalent influenza vaccine were established with the optimized in vitro micro-cytotoxicity test and dose-response (serum dilution vs absorbance) curves are presented for influenza virus A ~ Taiwan (Fig. 2). Neutralization of the influenza virus induced cytotoxicity by sera obtained after immunization with influenza vaccine is clearly demonstrated by
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with the trivalent embryonated egg derived influenza vaccine. Sera were obtained 7 days after booster immunization; dose: 0 /xg HA (zx); dose: 0.94 ~g HA (o); dose: 3.75 /xg HA (o); dose: 15/~g HA ( ~ ); and dose: 60/~g HA ( [] ). the course of the dose-response curves. The neutralization titers, defined as the reciprocal of the serum dilution giving a 50% reduction of the cytotoxic effect, increased from 320 (0.94/zg dose) to 1280 (60 /.~g dose). The low dose-response curve of the control group (0 ~ g dose) indicates that at low serum dilutions ( < 1/40) slight nonspecific interactions could occur. The relationship between neutralization (N) titers obtained with the in vitro neutralization test and the hemagglutination inhibition (HA1) titers obtained with the
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Fig. 4. Neutralization of the Chikungunya virus induced cytotoxic effect by sera obtained from rabbits immunized with UV-irradiated Chikungunya virus. P r e - i m m u n e serum ([]) and sera obtained 21 days after priming ( o , rabbit 1; o, rabbit 2) and 14 days after booster immunization (e, rabbit 1; *, rabbit 2) were investigated.
HAI-test is presented in Fig. 3. A linear relationship was found between N titers and HA1 titers (coefficient of correlation = 0.9562). The results obtained with the in vitro neutralization tests for influenza virus A / B e y i n g and B / Y a m agat a were comparable to the influenza virus A / T a i w a n results. For Chikungunya virus the neutralizing antibody response was investigated 21 days after priming and 14 days after a booster immunization with UV-irradiated Chikungunya virus (Fig. 4). The effect of a booster immunization is clearly visible; the neutralization titers increased from 480 and 960 (21 days after priming) to 3840 and 15,360 (14 days after booster immunization). The results presented for influenza viruses and Chikungunya virus demonstrate the broad applicability of in vitro micro-cytotoxicity tests in vaccine-efficacy studies and serological studies, i.e., the possibility of measuring antibody levels which neutralize biological agents.
Discussion
The in vitro micro-cytotoxicity tests described for the detection and quantitation of neutralizing antibodies to Shigella toxin, influenza viruses and Chikungunya virus demonstrate the broad applicability of these assays. The method can be easily
adapted to other biological agents, both toxins and viruses. The main modification comprises an adaptation of the system to the most sensitive adherent target cell for the biological agent under investigation. The in vitro micro-cytotoxicity tests therefore may be very useful for both vaccine-efficacy studies and serological studies. Furthermore, these tests may also be a handy tool for the screening and selection of specific antibodies. However, in this approach only neutralizing antibodies can be selected whereas for several applications, e.g., detection purposes, non-neutralizing antibodies may be suitable as well. Since in vitro micro-cytotoxicity tests are based on the specific cytotoxic properties of the biological agent under investigation, false positive responses caused by impurities in the biological agent preparation can be avoided. The in vitro micro-cytotoxicity test for the detection and quantitation of neutralizing antibodies is a very sensitive method. This is illustrated by the dose-response (serum dilution vs. absorbance) curves which were obtained. For antiChikungunya virus in particular steep dose-response curves are obtained (Fig. 4). A rapid decline of the dose-response curves within one step of a serial two-fold dilution was observed. The higher sensitivity of in vitro micro-cytotoxicity tests for neutralizing anti-viral antibodies in comparison with the tests for the determination of
163 neutralizing anti-toxin antibodies can be explained by the replication of viruses in target cells. Infection of a target cell by only one virus particle can result in cell death as a result of virus replication whereas in the case of toxins, many toxin molecules are required to obtain such a lethal effect. The potency of virulent virus particles to replicate in target cells suggests that further improvements in the sensitivity can be obtained if longer incubation times are used. Virus particles produced after first round of replication can only induce cytotoxicity again if long incubation times are used. The amplification obtained as a result of virus replication is the basis of the higher sensitivity of the system. Whether prolonged incubation times will be used for the determination of neutralizing anti-viral antibodies depends on the situation. The advantages of sensitivity and rapidity must be weighed against each other for each specific case. The optimized in vitro micro-cytotoxicity tests for the determination of neutralizing antibodies to Shigella toxin, influenza viruses and Chikungunya virus differ from each other with respect to (i) type of target cell, (ii) cell density, (iii) medium, and (iv) incubation times. As a result of these differences the maximum absorbance obtained in the dose-response curves (Figs. 1, 2, and 4) varied from 0.55 (anti-Chikungunya assay) to 1.60 (antiShigella toxin assay). Since the cause of the induced cytotoxicity is different for various biological agents, it is not surprising that the time at which cytotoxicity is observed can also be different. This may also be an explanation for the differences in the optimized incubation times, i.e., 48 h for Shigella toxin, 24 h for influenza viruses and 28 h for Chikungunya virus. For Shigella toxin, the cytotoxic effect is less pronounced if the incubation time is shortened. In the case of influenza viruses and Chikungunya virus shortening of the incubation time by only a few hours results in a sudden decrease in cytotoxicity. This indicates that for viruses at least one complete cycle of virus replication is required to obtain the desired cytotoxic effect. Since the presented in vitro micro-cytotoxicity tests gives information about the neutralizing capacity of antisera, these tests may be very useful
for serological studies and vaccine-efficacy studies. Although the neutralization data obtained are indicative of the protective capacity of the antisera, some reservations remain with respect to making absolute statements on the protective immunity in vivo to biological agents. Comparative studies between the in vitro micro-cytotoxicity tests and sensitive animal models should demonstrate if 'in vitro neutralization' correlates with 'humoral protection in vivo'. A good correlation between 'neutralization in vitro' and 'humoral protection in vivo' diminishes the necessity of using animal models in the evaluation of protective immunity to biological agents. The in vitro micro-cytotoxicity tests are rapid and performed in the wells of microtiter plates thereby facilitating the screening of large numbers of samples. Therefore, for reasons of efficiency, the use in vitro micro-cytotoxicity tests offers several advantages.
Acknowledgements The Shigella toxin and Chikungunya virus projects were sponsored by the Dutch Ministry of Defence. The influenza virus project was partly supported by S o l v a y / D u p h a r (Weesp, Netherlands). Dr. G. van Scharrenburg (Solvay/Duphar) is gratefully acknowledged for providing the influenza vaccine and influenza viruses.
References Bernstein, D.I., Kacica, M.A., McNeal, M.M., Schiff, G.M. and Ward, R.L. (1989) Local and systemic antibody response to rotavirus WC3 vaccine in adult volunteers. Antiviral Res. 12, 293. Boersma, W.J.A., Bogaerts, W.J.A., Bianchi, A.T.J. and Claassen, E. (1992)Adjuvant properties of stable water-inoil emulsions: evaluation of the experience with Specol. Forum in Immunology.Res. Immunol. 143, 503. Bokhout, B.A., Van Gaalen C., Van der Heijden J. (1981) A selected water-in-oil emulsion: compositionand usefulness as an immunological adjuvant. Vet. Immunol. Immunopathol. 2, 491. Conrath, T.B. (1972) Handbook of Microtiter Procedures. Dynatech Corporation, Cambridge, MA, p. 388. Davis, J.L., Hodge, H.M. and Campbell, W.E. (1971) Growth of Chikungunya virus in baby hamster kidney cell (BHK21-clone 13) suspension cultures. Appl. Microbiol. 21,338.
164 Donohue-Rolfe, A., Keusch, G.T., Edson, C., Thorley-Lawson, D. and Jacewicz, M. (1984) Pathogenesis of Shigella diarrhea, IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies. J. Exp. Med. 160, 1767. Gentry, M.K. and Dalrymple, J.M. (1980) Quantitative microtiter cytotoxicity assay for Shigella toxin. J. Clin. Microbiol. 12, 361. Keusch, G.T., Donohue-Rolfe, A., Jacewicz, M. and Kane, A.V. (1988) Shiga toxin: production and purification. Methods Enzymol. 165, 152. Knowlton, D.R., Spector, D.M. and Ward, R.L. (1991) Development of an improved method for measuring neutralizing antibody to rotavirus. J. Virol. Methods 33, 127. Matsumura, T., Shiraki, K., Hotta, S. and Sashikata, T. (1972) Release of Arboviruses from cells cultivated with low ionic strength media. Proc. Soc. Exp. Biol. Med. 141,599. Nakao, E. and Hotta, S. (1973) Immunogenicity of purified, inactivated chikungunya virus in monkeys. Bull. WHO 48, 559. Okuno, Y., Tanaka, K., Baba, K., Maeda, A., Kunita, N. and Ueda, S. (1990) Rapid focus reduction neutralization test
of influenza A and B viruses in microtiter system. J. Clin. Microbiol. 28, 1308. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76. Tomiyama, T., Sugano, T., Tani, S., Hosoda, K. and Matsumoto, Y. (1993) A microneutralization enzyme immunoassay for antibody to human cytomegalovirus. J. Immunol. Methods 159, 71. Wood, J.M., Oxford, J.S., Dunleavy, U., Newman, R.W., Major, D. and Robertson, J.S. (1989) Influenza A (H1N1) vaccine efficay in animal models is influenced by two amino acid substitutions in the hemagglutinin molecule. Virology 171, 214. Zegers, N.D., Claassen, E., Neelen, C., Mulder, E., Van Laar, J., Voorhorst, M.M., Berrevoets, C.A., Brinkmann, A.O., Van der Kwast, Th., Ruizeveld de Winter, J.A., Trapman, J.A. and Boersma, W.J.A. (1991) Epitope prediction and confimation for the human androgen receptor: Generation of monolconal antibodies for multi-assay performance following the synthetic peptide strategy. Biochem. Biophys. Acta 1073, 23-32.
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© 1993 ElsevierSciencePublishers B.V. All rights reserved0022-1759/93/$06.00
JIM06850
Rapid in vitro micro-cytotoxicity tests for the detection and quantitation of neutralizing antibodies to both viruses and toxins C. van de W a t e r a, E.A. van D u r a a, J.G.M.M. van der Stap a, R. Brands b and W.J.A. B o e r s m a .,a Department of Immunology and Medical Microbiology, T.N.O. Medical Biological Laboratory, P.O. Box 5815, 2280 HV Rijswijk, Netherlands, and b Solvay / Duphar, P.O. Box 900, 1380 DA Weesp, Netherlands (Received 8 June 1993, revised received 5 July 1993, accepted 6 July 1993)
A generally applicable method was developed for the rapid and quantitative detection of both toxinand virus neutralizing antibodies. The method was optimized for three different biological agents, i.e., Shigella toxin, influenza viruses (A/Beying, A / T a i w a n and B/Yamagata) and Chikungunya virus. The in vitro micro-cytotoxicity tests developed for the detection and quantitation of neutralizing antibodies are based on the inhibition of the virus- or toxin-induced cytotoxic effect by antibodies. As a result of the cytotoxicity, infected cells are no longer attached to the solid phase and can be easily removed. Thereafter, the proteins of the remaining living cells are stained. After removing the excess dye, the remaining dye is dissolved and the absorbance values are measured. The neutralization titers are determined from the absorbance values. Since the tests are performed in wells of microtiter plates, the in vitro micro-cytotoxicity tests are less laborious and consume less reagent in comparison with classical neutralization tests. Key words: Neutralizing antibody; Detection; Cytotoxicity; Protection; Immune response; Vaccine efficacy; Shigella toxin; Influenza virus; Chikungunya virus
Introduction
In order to estimate the efficacy of vaccines in protection against pathogens or toxins, it is usual to determine the ability of the evoked serum antibodies to neutralize cytopathogenic effects.
* Corresponding author. Tel.: +31 15 843188; Fax: +31 15 843989. Abbreviations: BCA, bicinchoninic)acid; BHK, baby hamster kidney; DMEM, Dulbecco's modification of Eagle's medium; ELISA, enzyme-linked immunosorbent assay; FRN, focus reduction neutralization; HA, hemagglutinin; HAl, hemagglutination inhibition; MDCK, Madin-Darby canine kidney; N, neutralization; pfu, plaque forming unit(s); PBS, phosphatebuffered saline; SRD, single radial diffusion.
Furthermore, detection and quantitation of the neutralizing antibody response may also be a valuable tool for the determination of the humoral immune status of humans or animals after exposure to biological agents. Consequently, the level of circulating antibodies with neutralizing capacity indicates the immune responsiveness of individuals a n d / o r the immunogenicity of the antigen. Classical methods, such as the conventional plaque reduction assays for antiviral antibodies, can be used for quantitation of the neutralizing antibody response but these methods are both expensive and time-consuming and the results are obtained by visual interpretation. The focus reduction neutralization (FRN) assays (Bernstein et al., 1989; Okuno et al., 1990) are
158
performed in 96-well tissue culture plates and are based on the detection and enumeration of foci stained by means of enzyme immunochemical techniques. These tests are less costly, less laborious and generally more reproducible. The results, however, are obtained after visual observation. Modified FRN assays, i.e., the micro-neutralization immunoassays (Knowlton et al., 1991; Tomiyama et al., 1993) combine the advantages of both FRN assays and ELISA. In micro-neutralization immunoassays the results are easily quantifiable because they are obtained by means of an ELISA reader instead of visual estimation. Therefore, the micro-neutralization immunoassay represents an improvement over the FRN-assay. However, both the FRN-assay and the microneutralization immunoassay require specific antibody preparations and are limited to the detection of neutralizing antiviral antibodies. In this paper in vitro micro-cytotoxicity tests for screening of neutralizing antibodies are described. These tests are based on the cytotoxicity assay as described for the quantitation of Shigella toxin (Gentry and Dalrymple, 1980). In comparison with the micro-neutralization immunoassay, the present in vitro micro-cytotoxicity tests have the advantage of (i) broad applicability, and (ii) ease of optimization for other biological agents. This means that the in vitro micro-cytotoxicity tests can be used for screening neutralizing antibodies to both viruses and toxins. Since specific antibody preparations are not required for the detection and quantitation of neutralizing antibodies, the in vitro micro-cytotoxicity tests can easily be broadened to other biological agents which have a cytotoxic effect on target cells growing on adherent monolayers. This is demonstrated in this paper by application of the in vitro micro-cytotoxicity tests for screening neutralizing antibodies to three totally different biological agents, i.e., Shigella toxin, influenza viruses and Chikungunya virus. Materials and methods
Vaccines, uiruses, toxin and cells Trivalent embryonated egg derived influenza vaccine (1992: A/Beying, A/Taiwan, B/Yamagata) and influenza viruses A/Beying, A / T a i w a n
and B/Yamagata were a kind gift of R. Brands and G. van Scharrenburg (Solvay/Duphar, Weesp, Netherlands). Chikungunya virus was kindly supplied by Y. Okuno (Research Institute for Microbial Diseases, Osaka University, Japan). Shigella toxin was produced as described by Keusch et al. (1988). The 60R strain of Shigella dysenteriae, being used for the production of Shigella toxin was a gift of A.D. O'Brien (Department of Microbiology, Uniformed Services of the Health Sciences, Bethesda, MD, USA). The baby hamster kidney cell line (BHK21 clone 13) was obtained from the American Type Culture Collection. The Madin-Darby canine kidney (MDCK) cell line was obtained from Solvay/ Duphar. The HeLa $3 cell line used was in stock in our laboratory.
Cell culture The culture conditions for target cells being used in the in vitro micro-cytotoxicity tests for measuring anti-Shigella toxin and anti-Chikunkunya neutralizing antibodies, i.e., HeLa ceils and BHK cells respectively, have been described elsewhere (Gentry and Dalrymple, 1980; Davis et al., 1971). Target cells used in the in vitro microcytotoxicity test for measuring anti-influenza virus neutralizing antibodies, i.e., MDCK cells, have been described by Wood et al. (1989). Cell cultures of BHK, MDCK and HeLa cells were maintained in 25 cm 2 culture flasks at 37°C in a 5% CO 2 atmosphere in complete RPMI, serum-free EpiSerf medium and Dulbecco's modification of Eagle's medium (DMEM) with Ham's F-10 respectively. Complete RPMI medium consisted of RPMI 1640 medium with 106 IU penicillin/ml, 10 mg streptomycin/ml, 5 x 10 -5 M/3-mercaptoethanol and 10% fetal calf serum; serum-free EpiSerf medium was supplemented with 1.5 mg NaHCO3/ml, 500 IU penicillin/ml, 500 IU streptomycin/ ml and 0.5 mg glutamine/ml; D M E M / F 10 medium, 95 : 100 (v/v) was supplemented with 10% fetal calf serum, 110 IU penicillin/ml, 110 IU streptomycin/ml, 0.17 mg glutamine/ml and 2.3/zg hypoxanthine/ml. Cells were passaged every 5-7 days by the addition of trypsin-EDTA, passed into new 25 cm 2 culture flasks (2 x 10 4 BHK cells, 5 × 104
159 HeLa cells and 8 × 104 MDCK cells respectively) and incubated at 37°C under 5% CO 2 atmosphere. Sera The sera screened for neutralizing antibodies were obtained from mice and rabbits immunized with toxin- and virus-derived immunogens. Trivalent embryonated egg derived influenza vaccine (Influvac, Solvay/Duphar), inactivated Chikungunya virus and inactivated Shigella toxin were used as immunogens. Chikungunya virus was inactivated by UV irradiation as described by Nakoa and Hotta (1973). A single low pressure mercury vapour lamp (Phillips, 15 W, TUV) with an incidence fluence rate of 0.604 W / m 2 at 254 nm was used for 10 min to obtain complete inactivation of Chikungunya virus. Shigella toxin was inactivated by a 30 h treatment with 0.1% formaldehyde as described by Donohue-Rolfe et al. (1984). Groups of 12-week-old female B A L B / c mice were injected subcutaneously with influenza vaccine (0.94-60/~g), and intraperitoneally with inactivated Shigella toxin (50 /zg) and inactivated Chikungunya virus (50 /zg) respectively. Rabbits were injected intradermally with inactivated Chikungunya virus (200 Izg). Influenza vaccine was used without adjuvant. For the immunization of mice the Chikungunya virus and Shigella toxin preparations were emulsified in 'specol', i.e., a water-in-oil suspension (Bokhout et al., 1981; Boersma et al., 1992); booster immunizations were given 21 days after priming. For the immunization of rabbits the Chikungunya virus preparation was emulsified in complete Freund's adjuvant for priming and in incomplete Freund's adjuvant for booster injections. The booster injections were given 28 days after priming. The amounts of influenza vaccine were based on hemagglutin antigen as determined with the single radial diffusion (SRD) test; the amounts of inactivated Chikungunya virus and inactivated Shigella toxin were based on the results obtained with the BCA protein determination as described by Smith et al. (1985). Monoclonal antibodies Monoclonal antibodies specific for Shigella toxin and Chikungunya virus were produced by
hybridomas obtained from the fusion of the mouse SP2/0 plasmacytoma cell line with spleen ceils from BALB/c mice which were immunized with inactivated Shigella toxin or inactivated Chikungunya virus. The procedure followed was identical to the procedure described by Zegers et al. (1991) for the preparation of monoclonal antibodies to a~-antitrypsin. Plaque test The plaque test for Chikungunya virus was performed in an analogous manner to the procedure described by Matsumura et al. (1972). BHK monolayers were incubated with 100 /zl volumes of Chikungunya virus dilutions at 37°C, 5% CO 2 for 1 h. Overlay medium (RPMI containing 1.6% agarose) was added. After 3 days incubation at 37°C, 5% CO 2 the cultures were stained and fixed with a solution containing 0.2% crystal violet and 0.8% ammonium oxalate. After removing of the overlay medium and washing of the stained ceils with demineralized water, the plaques were counted. Hemagglutination inhibition test Hemagglutination inhibition (HAD tests were performed in microtiter plates by standard methods. After preincubation of serum dilutions with 2 HA units of influenza virus A / T a i w a n (50 /xl) for 1 h at 37°C, 100/~1 volumes of a 0.5% chicken erythrocyte suspension in physiological saline solution were added. The microtiter plate was incubated for 1 h at 4°C and the hemagglutination titer, defined as the reciprocal of the serum dilution at which agglutination just disappeared, was read by eye. To eliminate serum factors which could inhibit non-specific agglutination, the sera were pretreated with receptor-destroying enzyme (Conrath, 1972). In vitro micro-cytotoxicity test MDCK cells with supplemented serum-free EpiSerf medium, BHK cells with complete RPMI medium and HeLa ceils with supplemented DMEM/F-10 medium were used in the in vitro micro-cytotoxicity tests for quantitation of influenza virus, Chikungunya virus and Shigella toxin respectively. The in vitro micro-cytotoxicity tests were based on the cytotoxicity tests previ-
160 ously described for the quantitation of Shigella toxin (Gentry and Dalrymple, 1980). For the detection and quantitation of influenza virus-, Chikungunya virus- and Shigella toxin neutralizing-antibodies, preincubations of virus or toxin with serum/supernatant dilutions were performed. Optimized in vitro micro-cytotoxicity tests for measuring neutralizing antibodies were performed as follows. Serial two-fold dilutions of the sera under investigation were prepared in the wells of a 96-well microtiter plate (Costar, Cambridge, MA, USA) using supplemented EpiSerf medium, complete RPMI and supplemented DMEM/F-10 medium as dilution buffers for the influenza virus, Chikungunya virus and Shigella toxin neutralization assay respectively. Influenza virus A / T a i w a n (50 ~l 40 HA U / m l ) , Chikungunya virus (50 /~1 480 p f u / m l ) and Shigella toxin (50/.d 126 ng/ml) respectively were added to 50 ~l volumes of the serum dilutions. After incubation for 1 h at 37°C, 100/~l volumes of freshly trypsinized cells resuspended in medium (4 × 105 M D C K / m l EpiSerf; 2 × 105 B H K / m l RPMI; 1 × 105 H e L a / m l DMEM/F-10) were added. After incubation at 37°C, 5% CO 2 ('Shigella toxin' 48 h; 'Chikungunya virus' 28 h and 'influenza viruses' 24 h) the medium was aspirated. The wells of the microtiter plates were washed twice with 200 ~l of PBS. The residual living cells were fixed and their proteins stained with 50 ~l of a solution of 0.5% 2.00
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Amido black in methanol/demineralized water/ acetic acid, glacial (5 : 5 : 1). After 20 min incubation at room temperature the staining solution was removed and the wells were subsequently washed with destaining solution, i.e., methanol/ demineralized water/acetic acid, glacial (5 : 5 : 1) and demineralized water. The protein-associated dye was dissolved in 100 /~I of PBS/ethanol (1:1) and the absorbance values were measured at 580 nm using an ELISA reader (Flow Titertek Multiskan 8 channel reader, Flow Laboratories, Irvine, Scotland). The neutralization titers, defined as the reciprocal of the serum dilutions giving a 50% reduction of the cytotoxic effect, were calculated.
Results
Optimum conditions for in vitro micro-cytotoxicity tests In vitro micro-cytotoxicity tests were developed for both Shigella toxin, influenza viruses (A/Beying, A / T a i w a n and B/Yamagata) and Chikungunya virus. Initially the optimal cell-culture conditions for Shigella toxin, influenza virus and Chikungunya virus sensitive cells, i.e., HeLa, MDCK and BHK cells respectively, were established. The cytotoxicity tests were optimized for cell concentration and incubation time. For Shigella toxin, influenza viruses and Chikungunya virus, the optimized incubation times were established to be 48 h, 24 h and 28 h respectively. In case of Chikungunya virus, the sensitivity of the cytotoxicity test could be easily improved by increasing the incubation time to 48 h and lowering the cell concentration to 0.5 × 105 BHK cells/ml. This phenomenon must be explained by infection of BHK cells with virus particles produced after the first round of replication. However, the sensitivity of the influenza virus cytotoxicity tests could not be further increased by prolonging the incubation time and lowering the cell concentration. To validate the cytotoxicity test for Chikungunya virus, this assay was compared with a classical plaque test. The amount of Chikungunya virus resulting in a 50% cytotoxic effect in the cytotoxicity test corresponded to six plaque forming units (pfu) in the classical plaque test. This
161 suggested that the sensitivities of both tests were similar. The 50% cytotoxic value in the cytotoxicity test indicated that 50% of the cells were infected, i.e., 1 x 104 cells. Extrapolation of these results to the classical plaque test showed that the number of pfu measured (i.e., six) was rather low in comparison with the maximum number of pfu which could be obtained (i.e., 104). The optimal virus and toxin concentrations in the in vitro micro-cytotoxicity tests for the detection and quantitation of neutralizing antibodies, i.e., defined as the virus or toxin concentrations at which 90% of the ceils were detached from the solid-phase, were established by means of doseresponse curves in the cytotoxicity tests.
In vitro micro-cytotoxicity tests The in vitro micro-cytotoxicity tests were very useful for the development of immunochemical methods, in particular with respect to screening and selection of polyclonal and monoclonal antibodies. The in vitro micro-cytotoxicity tests were successfully applied for the selection of neutralizing monoclonal antibodies to both Shigella toxin and Chikungunya virus. A dose-response curve of a Shigella toxin neutralizing monoclonal antibody is presented in Fig. 1. Since non-specific interactions caused by normal mouse serum or monoclonal antibodies to other biological agents were not observed (results not shown), the dose-response dependency presented in Fig. 1 could be completely attributed to the specific neutralizing capacity of the monoclonal antibody. The protective capacity of this monoclonal antibody is indicated by the high neutralization titer (640 for Shigella toxin 126 n g / m l ) . The application of this monoclonal antibody in immunochemical methods for the determination of Shigella toxin will be the subject of further investigation. Influenza virus neutralizing antibodies in sera obtained from mice immunized with trivalent influenza vaccine were established with the optimized in vitro micro-cytotoxicity test and dose-response (serum dilution vs absorbance) curves are presented for influenza virus A ~ Taiwan (Fig. 2). Neutralization of the influenza virus induced cytotoxicity by sera obtained after immunization with influenza vaccine is clearly demonstrated by
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with the trivalent embryonated egg derived influenza vaccine. Sera were obtained 7 days after booster immunization; dose: 0 /xg HA (zx); dose: 0.94 ~g HA (o); dose: 3.75 /xg HA (o); dose: 15/~g HA ( ~ ); and dose: 60/~g HA ( [] ). the course of the dose-response curves. The neutralization titers, defined as the reciprocal of the serum dilution giving a 50% reduction of the cytotoxic effect, increased from 320 (0.94/zg dose) to 1280 (60 /.~g dose). The low dose-response curve of the control group (0 ~ g dose) indicates that at low serum dilutions ( < 1/40) slight nonspecific interactions could occur. The relationship between neutralization (N) titers obtained with the in vitro neutralization test and the hemagglutination inhibition (HA1) titers obtained with the
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hemagglutination inhibition (HAD test for influenza virus A/Taiwan.
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Fig. 4. Neutralization of the Chikungunya virus induced cytotoxic effect by sera obtained from rabbits immunized with UV-irradiated Chikungunya virus. P r e - i m m u n e serum ([]) and sera obtained 21 days after priming ( o , rabbit 1; o, rabbit 2) and 14 days after booster immunization (e, rabbit 1; *, rabbit 2) were investigated.
HAI-test is presented in Fig. 3. A linear relationship was found between N titers and HA1 titers (coefficient of correlation = 0.9562). The results obtained with the in vitro neutralization tests for influenza virus A / B e y i n g and B / Y a m agat a were comparable to the influenza virus A / T a i w a n results. For Chikungunya virus the neutralizing antibody response was investigated 21 days after priming and 14 days after a booster immunization with UV-irradiated Chikungunya virus (Fig. 4). The effect of a booster immunization is clearly visible; the neutralization titers increased from 480 and 960 (21 days after priming) to 3840 and 15,360 (14 days after booster immunization). The results presented for influenza viruses and Chikungunya virus demonstrate the broad applicability of in vitro micro-cytotoxicity tests in vaccine-efficacy studies and serological studies, i.e., the possibility of measuring antibody levels which neutralize biological agents.
Discussion
The in vitro micro-cytotoxicity tests described for the detection and quantitation of neutralizing antibodies to Shigella toxin, influenza viruses and Chikungunya virus demonstrate the broad applicability of these assays. The method can be easily
adapted to other biological agents, both toxins and viruses. The main modification comprises an adaptation of the system to the most sensitive adherent target cell for the biological agent under investigation. The in vitro micro-cytotoxicity tests therefore may be very useful for both vaccine-efficacy studies and serological studies. Furthermore, these tests may also be a handy tool for the screening and selection of specific antibodies. However, in this approach only neutralizing antibodies can be selected whereas for several applications, e.g., detection purposes, non-neutralizing antibodies may be suitable as well. Since in vitro micro-cytotoxicity tests are based on the specific cytotoxic properties of the biological agent under investigation, false positive responses caused by impurities in the biological agent preparation can be avoided. The in vitro micro-cytotoxicity test for the detection and quantitation of neutralizing antibodies is a very sensitive method. This is illustrated by the dose-response (serum dilution vs. absorbance) curves which were obtained. For antiChikungunya virus in particular steep dose-response curves are obtained (Fig. 4). A rapid decline of the dose-response curves within one step of a serial two-fold dilution was observed. The higher sensitivity of in vitro micro-cytotoxicity tests for neutralizing anti-viral antibodies in comparison with the tests for the determination of
163 neutralizing anti-toxin antibodies can be explained by the replication of viruses in target cells. Infection of a target cell by only one virus particle can result in cell death as a result of virus replication whereas in the case of toxins, many toxin molecules are required to obtain such a lethal effect. The potency of virulent virus particles to replicate in target cells suggests that further improvements in the sensitivity can be obtained if longer incubation times are used. Virus particles produced after first round of replication can only induce cytotoxicity again if long incubation times are used. The amplification obtained as a result of virus replication is the basis of the higher sensitivity of the system. Whether prolonged incubation times will be used for the determination of neutralizing anti-viral antibodies depends on the situation. The advantages of sensitivity and rapidity must be weighed against each other for each specific case. The optimized in vitro micro-cytotoxicity tests for the determination of neutralizing antibodies to Shigella toxin, influenza viruses and Chikungunya virus differ from each other with respect to (i) type of target cell, (ii) cell density, (iii) medium, and (iv) incubation times. As a result of these differences the maximum absorbance obtained in the dose-response curves (Figs. 1, 2, and 4) varied from 0.55 (anti-Chikungunya assay) to 1.60 (antiShigella toxin assay). Since the cause of the induced cytotoxicity is different for various biological agents, it is not surprising that the time at which cytotoxicity is observed can also be different. This may also be an explanation for the differences in the optimized incubation times, i.e., 48 h for Shigella toxin, 24 h for influenza viruses and 28 h for Chikungunya virus. For Shigella toxin, the cytotoxic effect is less pronounced if the incubation time is shortened. In the case of influenza viruses and Chikungunya virus shortening of the incubation time by only a few hours results in a sudden decrease in cytotoxicity. This indicates that for viruses at least one complete cycle of virus replication is required to obtain the desired cytotoxic effect. Since the presented in vitro micro-cytotoxicity tests gives information about the neutralizing capacity of antisera, these tests may be very useful
for serological studies and vaccine-efficacy studies. Although the neutralization data obtained are indicative of the protective capacity of the antisera, some reservations remain with respect to making absolute statements on the protective immunity in vivo to biological agents. Comparative studies between the in vitro micro-cytotoxicity tests and sensitive animal models should demonstrate if 'in vitro neutralization' correlates with 'humoral protection in vivo'. A good correlation between 'neutralization in vitro' and 'humoral protection in vivo' diminishes the necessity of using animal models in the evaluation of protective immunity to biological agents. The in vitro micro-cytotoxicity tests are rapid and performed in the wells of microtiter plates thereby facilitating the screening of large numbers of samples. Therefore, for reasons of efficiency, the use in vitro micro-cytotoxicity tests offers several advantages.
Acknowledgements The Shigella toxin and Chikungunya virus projects were sponsored by the Dutch Ministry of Defence. The influenza virus project was partly supported by S o l v a y / D u p h a r (Weesp, Netherlands). Dr. G. van Scharrenburg (Solvay/Duphar) is gratefully acknowledged for providing the influenza vaccine and influenza viruses.
References Bernstein, D.I., Kacica, M.A., McNeal, M.M., Schiff, G.M. and Ward, R.L. (1989) Local and systemic antibody response to rotavirus WC3 vaccine in adult volunteers. Antiviral Res. 12, 293. Boersma, W.J.A., Bogaerts, W.J.A., Bianchi, A.T.J. and Claassen, E. (1992)Adjuvant properties of stable water-inoil emulsions: evaluation of the experience with Specol. Forum in Immunology.Res. Immunol. 143, 503. Bokhout, B.A., Van Gaalen C., Van der Heijden J. (1981) A selected water-in-oil emulsion: compositionand usefulness as an immunological adjuvant. Vet. Immunol. Immunopathol. 2, 491. Conrath, T.B. (1972) Handbook of Microtiter Procedures. Dynatech Corporation, Cambridge, MA, p. 388. Davis, J.L., Hodge, H.M. and Campbell, W.E. (1971) Growth of Chikungunya virus in baby hamster kidney cell (BHK21-clone 13) suspension cultures. Appl. Microbiol. 21,338.
164 Donohue-Rolfe, A., Keusch, G.T., Edson, C., Thorley-Lawson, D. and Jacewicz, M. (1984) Pathogenesis of Shigella diarrhea, IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies. J. Exp. Med. 160, 1767. Gentry, M.K. and Dalrymple, J.M. (1980) Quantitative microtiter cytotoxicity assay for Shigella toxin. J. Clin. Microbiol. 12, 361. Keusch, G.T., Donohue-Rolfe, A., Jacewicz, M. and Kane, A.V. (1988) Shiga toxin: production and purification. Methods Enzymol. 165, 152. Knowlton, D.R., Spector, D.M. and Ward, R.L. (1991) Development of an improved method for measuring neutralizing antibody to rotavirus. J. Virol. Methods 33, 127. Matsumura, T., Shiraki, K., Hotta, S. and Sashikata, T. (1972) Release of Arboviruses from cells cultivated with low ionic strength media. Proc. Soc. Exp. Biol. Med. 141,599. Nakao, E. and Hotta, S. (1973) Immunogenicity of purified, inactivated chikungunya virus in monkeys. Bull. WHO 48, 559. Okuno, Y., Tanaka, K., Baba, K., Maeda, A., Kunita, N. and Ueda, S. (1990) Rapid focus reduction neutralization test
of influenza A and B viruses in microtiter system. J. Clin. Microbiol. 28, 1308. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76. Tomiyama, T., Sugano, T., Tani, S., Hosoda, K. and Matsumoto, Y. (1993) A microneutralization enzyme immunoassay for antibody to human cytomegalovirus. J. Immunol. Methods 159, 71. Wood, J.M., Oxford, J.S., Dunleavy, U., Newman, R.W., Major, D. and Robertson, J.S. (1989) Influenza A (H1N1) vaccine efficay in animal models is influenced by two amino acid substitutions in the hemagglutinin molecule. Virology 171, 214. Zegers, N.D., Claassen, E., Neelen, C., Mulder, E., Van Laar, J., Voorhorst, M.M., Berrevoets, C.A., Brinkmann, A.O., Van der Kwast, Th., Ruizeveld de Winter, J.A., Trapman, J.A. and Boersma, W.J.A. (1991) Epitope prediction and confimation for the human androgen receptor: Generation of monolconal antibodies for multi-assay performance following the synthetic peptide strategy. Biochem. Biophys. Acta 1073, 23-32.