Antibodies by Enzyme-Linked Immunosorbent Assay

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1983, p /83/ $02.00/0 Vol. 18, No. 2 Determination of Herpes Simplex Virus Type-Specific Antibodies by Enzyme-Linked Immunosorbent Assay R. MARIE COLEMAN,'* LENORE PEREIRA,2 PAULA D. BAILEY,' DALE DONDERO,2 CAROLYN WICKLIFFE,1 AND ANDRE J. NAHMIAS' Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia and Viral and Rickettsial Disease Laboratory, State of California Department of Health Services, Berkeley, California Received 29 November 1982/Accepted 15 April 1983 We determined type-specific antibodies to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by an indirect enzyme-linked immunosorbent assay, using as antigens HSV-1 glycoprotein gc-1 and a HSV-2-specific polypeptide purified on affinity columns of monoclonal antibodies. All sera were initially screened for HSV antibodies by the enzyme-linked immunosorbent assay with a pool of Triton X-100-extracted antigens of HSV-1- and HSV-2-infected HEp-2 cells. The titer of HSV antibodies was predicted from a linear regression curve based on the absorbance of the initial 1:50 serum dilution. The sensitivity and specificity of the screening assay and of the assay for type-specific antibodies were established. Numerous methods for detecting antibodies to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) have been reported (for a review, see reference 19). However, the delineation of specific HSV-1 and HSV-2 antibodies has been difficult because of the strong cross-reactivity between the two viruses (11, 21). Several techniques have been applied to the problem of differentiating human sera reactive with HSV-1, HSV-2, or both. These include micro-neutralization tests (13, 14, 20), fluorescent-antibody tests (25), antibody-complement cytolysis (24), hemagglutination-inhibition assays (2, 3, 21, 22), enzyme-linked immunosorbent assays (ELISA) (26), and radioimmunoassay (6, 7, 9, 15). The optimal way would be to test the reactivity of sera with purified type-specific antigens. This approach became technically possible with the development of monoclonal antibodies to several HSV-1 and HSV-2 proteins (17, 18). Thus, monoclonal antibodies have been used to prepare immunoaffinity columns for the purification of HSV glycoproteins gd (5) and HSV-1 glycoprotein gc (1). In addition, monoclonal antibody H379, which has been found to react type specifically with HSV isolates (17, 18), has been used to purify a HSV-2-specific polypeptide (L. Pereira and D. Dondero, manuscript in preparation). In this report, we describe two types of enzyme immunoassays. The first is for the detection and titration of total antibodies to HSV-1 and HSV-2 with antigens prepared by extracting infected cells with Triton X-100. The second assay is for HSV type-specific antibody differentiation with the immunoaffinity-purified HSV specific glycoprotein gc and a type-specific HSV-2 polypeptide. MATERIALS AND METHODS Patient population. Sera were obtained from individuals with clinically manifest and culturally proven primary or recurrent infections with HSV-1 or HSV-2 or both. All viral isolates were typed as HSV-1 or HSV-2 by direct immunofluorescence (12). HSV infections were classified as primary infections if the individual had no preexisting neutralizing antibody to HSV-1 or HSV-2 and seroconversion was documented. For certain studies, we used sera which had already been tested by neutralization assays for other purposes and a collection of sera from children 2 to 12 years of age. Crude antigen preparations. For the routine screening of sera for HSV antibodies, antigens were prepared from HEp-2 cells infected with HSV-1 (F strain) and HSV-2 (G strain). After 4+ cytopathic effect (80 to 90%6 of the cells were rounded) was observed (24 to 48 h), the cells were harvested, sonicated in ph 8.6 Trisglycine buffer containing 2% Triton X-100 (27), clarified by centrifugation, and stored at -70 C. Control antigens of uninfected HEp-2 cells were prepared by the same protocol. Purification of HSV proteins. The properties of monoclonal antibodies HCI and H379 used to prepare immunoaffinity columns were reported previously (17, 18). The procedures for the preparation of the affinity columns and elution of the proteins were as previously described (1, 8). ELISA. ELISA (28) was performed for the detection of immunoglobulin G (IgG) antibodies to the Triton X- 100-extracted HSV antigens and for the determination of type-specific antibodies. The buffer used for the dilution of the Triton X-100-extracted antigen was 0.05 M carbonate (ph 9.6); for the dilution of purified proteins, phosphate-buffered saline (ph 7.4) was used;

2 288 COLEMAN ET AL. for the dilution of sera, phosphate-buffered saline with 1% bovine serum albumin and 0.05% Tween 20 was used; for washing the microplates, phosphate-buffered saline with 0.05% Tween 20 was used. For the screening of sera for total HSV antibodies, 100,ul of a pooled antigen of HSV-1 and HSV-2 or control antigen (1:1,600) was used to coat the wells of Immulon II polystyrene plates (Dynatech Laboratories, Inc., Alexandria, Va.). For the determination of type-specific antibodies, 50,ul of gc-1 (1:100 dilution) and the HSV- 2-specific polypeptide (1:50) was adsorbed to the Immulon II plates. All antigen-coated plates were incubated overnight at 4 C before use. The antigen-coated plates were washed three times (3 min each), and 100,ul of a 1:50 dilution of serum was added to duplicate wells of each antigen and incubated for 2 h at 37 C. The plates were washed, and 100,ul of a goat antihuman IgG peroxidase conjugate (Miles Laboratories, Inc., Elkhart, Ind.) was added and incubated for 45 min at 37 C. After washing the plates, we added 100,u1 of ph 6.0 citrate-buffered substrate (40 mg of o- phenylenediamine-2hcl and 0.05% H202 in 100 ml of substrate buffer of 7.74 g of citric acid and g of Na2HPO4 per liter of water). The reaction was stopped with 50,ul of 4 N H2SO4 after 10 min. The absorbance at 492 nm of each well was measured with an automatic plate reader (Titertek Multiskan; Flow Laboratories, Inc., McLean, Va.). The plate reader was interfaced with an Apple II Plus computer for the calculation of the mean and standard deviation of the absorbance for each serum. With these assays, the 1:50 dilution of serum was considered positive if the difference between the mean of the absorbance with the HSV-infected cell-extracted antigen or purified protein and that of the control antigen was >0.20. A total of 52 sera were titrated against the HSV- 1/HSV-2 combination antigen, and a linear regression curve was established by plotting the absorbance (492 nm) of the 1:50 dilution versus the final titer (r = 0.948). This linear regression curve was then used to predict the titer of HSV antibodies in the test sera based on the absorbance of the 1:50 dilution. The predicted titer analyses with the linear regression curve were also computerized (details of the computer programs are available from R. M. Coleman). To ensure the standardization of the assays, we included three control sera in each group of samples. These controls were a high-positive serum (titer of.1:12,800), low-positive serum (titer of 1:800), and a negative serum. The coefficient of variation for each control serum was calculated as the standard deviation expressed as a percentage of the mean. Microneutralzation tests. The procedures used for the microneutralization tests and the calculation of the neutralizing potency (pn) have been previously described (14, 16). A serum was labeled as type 1 if the pn difference was 20.5, type 2 if the pn difference was <0.05, and the intermediate (or dual) if the pn difference was between 0.05 and 0.5. The determination of type-specific antibodies by ELISA with affinity-purified antigens was compared with these previous determinations based on a calculation of the pn. RESULTS Screening assay. The results obtained when the screening assay was used to test 179 sera of J. CLIN. MICROBIOL. patients with various types of primary or recurrent HSV-1 or HSV-2 infections are given in Table 1. The specificity of the assay is shown by the negative results obtained from the sera of 43 individuals with no history or clinical evidence of HSV infection. ELISA was found to have increased sensitivity, at a starting serum dilution of 1:50, compared with the microneutralization test, at a starting dilution of 1:10. A total of 75 sera were positive for HSV antibodies by both tests, and 62 were negative by both tests. None was positive by the microneutralization assay and negative by ELISA, but 21 were negative by the microneutralization assay and positive by ELISA. It is unlikely that the positive results of these 21 sera that were negative by microneutralization represent false-positive reactions. Nine of these specimens were acute sera from individuals with primary genital HSV infections, five were acute sera from patients with herpes encephalitis, and six were acute sera from neonates with HSV. The remaining ELISA-positive neutralization-negative serum was from a patient who, although HSV culture negative from a suspect genital lesion, had later sera that were positive for HSV antibodies by both tests. Five of these sera had previously been found to be HSV-antibody positive by indirect hemagglutination tests (21). Six of the sera were also tested by antibody-dependent cellular cytotoxicity (23) and found to be positive. The screening assay was found to have a high degree of reproducibility. Based on the results of 11 assays, the mean specific absorbance of the TABLE 1. Screening assay of sera from patients with clinically manifest HSV-1 and HSV-2 infections and individuals with no evidence of HSV infection No. positive/ Sera from individuals with: no. tested in screening assay HSV-1 infections Herpes encephalitis... 24/24 Recurrent orolabial... 10/10 Primary genital Acute... 1/9 Convalescent... 14/14 HSV-2 infections Recurrent genital... 70/70 Primary genital Acute... 3/21 Convalescent... 31/31 No history or clinical evidence of HSV infection... 0/43

3 VOL. 18, 1983 HSV TYPE-SPECIFIC ANTIBODIES BY ELISA 289 TABLE 2. Correlation of predicted titer with actual titer Serum OD' at l:so Predicted Actual SerumOD' at 1:50 titer" titer :3,200 1:3, :6,400 1:6, A :12,800 1:12, B :12,800 1:25, A :400 1: A 1.9 1:6,400 1:6, :6,400 1:6, A 1.2 1:800 1: A 1.9 1:6,400 1:12, A 1.5 1:1,600 1:1, :3,200 1:6, :6,400 1:6, A 1.5 1:1,600 1:1, A :12,800 1:12, A 1.8 1:6,400 1:6, A 1.7 1:3,200 1:6,400 G :200 1:200 P :100 1:100 G :200 1: :3,200 1:6,400 a OD, Optical density. b Linear regression titers highly correlated, r = and actual titers were high-positive serum was found to be , that of the medium-positive serum was , and that of the negative serum was The coefficient of variation for these control sera ranged from 3 to 6.8%. Titer prediction from the linear regression curve was also shown to have a good accuracy index. As verified by the actual titration of 20 sera, predicted titers and actual titers were noted to be highly correlated (r = 0.974; Table 2). Assay for antibodies to purified HSV-1 and HSV-2 proteins. We tested the sera of 73 patients with primary, recurrent, or dual HSV infections by ELISA, using the affinity-purified HSV-1 and HSV-2 antigens (Table 3). We found generally good agreement between the virus type(s) isolated from the patients and the serological responses to the purified antigens. The major exception was 1 of the 36 sera from patients with recurrent genital HSV-2 infections that failed to react with the HSV-2 purified protein. Five of twenty sera obtained within 2 to 3 weeks of onset from individuals with primary HSV-2 infections failed to react with either purified protein. This may be due to a delay in the production of antibodies to the purified HSV-2 protein, as suggested by the absence of detectable antibodies to either the HSV-1 or HSV-2 purified antigens in sera obtained within the first 7 to 14 days after onset from patients with primary HSV-1 or HSV-2 infections (data not shown). When children between 2 and 12 years of age are infected by HSV, it is almost always HSV-1 (14). To ascertain further the specificity of the reactions with the HSV-1 and HSV-2 purified proteins, we first tested the sera of 127 children in this age group by the screening ELISA for HSV antibodies; sera positive by this assay were then tested for reactivity with the purified proteins. Thirty-seven (29%) of the sera were found to have HSV antibodies; 34 of 37 reacted only with gc-1, none reacted with only the HSV-2- specific protein, and 1 of 37 reacted with both type-specific antigens. This serum, which apparently contained dual antibodies, was absorbed with HSV-1- and HSV-2-infected HEp-2 cells and retested. Absorption confirmed the results obtained with the purified proteins. The presence of HSV antibodies, detected by the screening assay in the two sera which were negative with both purified proteins, was confirmed by antibody-dependent cellular cytotoxicity (23). A comparison of the determination in 42 sera of type-specific antibodies by ELISA, using the purified HSV-1 and HSV-2 proteins, and by the microneutralization tests is given in Table 4. ELISA permitted antibody type differentiation in six sera which could not be differentiated by microneutralization tests. One of the sera classified as dual by microneutralization was found to be reactive only with the HSV-2 protein. In addition, type-specific antibodies in two sera nonreactive with the purified proteins were not identified by the neutralization assay. ELISA and microneutralization tests yielded similar results in 32 of the sera: 14 HSV-1, 12 HSV-2, and 4 dual. DISCUSSION Using antigens prepared from Triton X-100- extracted HSV-infected cells, ELISA has been found to be a sensitive and specific test for the determination of total HSV antibodies. Extending the application of this assay by other workers (4, 5, 10, 26, 27), we have found that, by automation and computerization, it is possible to screen and predict the titer of as many as 150 to 200 sera per day. The absorbance of the ELISA reaction with a very small volume of serum at TABLE 3. Assay for type-specific antibodies No. No. of sera positive HSV infection *paof with: Type 1 Type 2 Type 1/ tients only only type 2 Primary genital HSV-1 ga Primary genital HSV-2 20a Recurrent genital HSV Dual HSV-1 and HSV a The sera were taken 2 to 3 weeks after onset.

4 290 COLEMAN ET AL. J. CLIN. MICROBIOL. TABLE 4. Comparison of HSV type-specific antibody determinations by ELISA and microneutralization tests No. of sera determined by microneutralization test ELISA with pna of: No. of sera that were neutralization negative > <0.05 CDb HSV-1 positive HSV-2 positive HSV-1/HSV-2 positive HSV-1/HSV-2 negative HSV-1 isolated HSV-2 isolated a pn, Neutralizing potency difference between HSV-1 and HSV-2. The pns of HSV-1 and HSV-2 were: >0.5 = HSV-1; 0.05 to 0.5 = dual; <0.05 = HSV-2. b CD, Cannot determine, as the pn cannot be calculated when the neutralization titers are low. one dilution (1:50) can be used to predict the titer of HSV antibodies. ELISA with affinity-purified HSV-1 and HSV-2 proteins has also been found to provide a sensitive and specific method for determining type-specific antibodies in sera. Our results confirm and extend the studies with radioimmunoassay using purified gc-1 recently reported by Arvin et al. (1). Other procedures for the determination of type-specific antibodies include the absorption of sera with heterologous HSV antigen (2, 3, 6, 9, 15, 21), the blocking of cross-reactive antigenic sites with heterologous antibody (26), and the immunoprecipitation of HSV-specific glycoproteins with human sera followed by polyacrylamide gel electrophoresis (7). Besides problems with incomplete absorption, these methods are not readily applicable for routine assays with large numbers of sera. In conclusion, we have demonstrated the use of ELISA for predicting titers with one serum dilution and for determining antibodies to certain purified HSV proteins. We are currently extending these data by determining antibody titers in various immunoglobulin subclasses to other purified HSV proteins. ACKNOWLEDGMENTS We thank Tom Spira, Centers for Disease Control, Atlanta, Ga., for performance of the antibody-dependent cellular cytotoxicity assay. This work was supported by Program Project Grant 1-POl- AX and Training Grant DE from the National Institutes of Health. LITERATURE CITED 1. Arvin, A. M., C. M. Koropchak, A. S. Yeager, and L. Pereira Detection of type-specific antibody to herpes simplex virus type 1 by radioimmunoassay with herpes simplex virus type 1 glycoprotein C with monoclonal antibody. Infect. Immun. 40: Back, A. F., and N. J. Schmidt Typing Herpesvirus hominis antibodies and isolates by inhibition of the indirect hemagglutination reaction. Appl. Microbiol. 28: Bernstein, M. T., and J. A. Stewart Method for typing antisera to Herpesvirus hominis by indirect hemagglutination inhibition. Appl. Microbiol. 21: Cremer, N. E., C. K. Cossen, C. V. Hanson, and G. R. Shell Evaluation and reporting of enzyme immunoassay determinations of antibody to herpes simplex virus in sera and cerebrospinal fluid. J. Clin. Microbiol. 15: Denoyel, G. A., A. Gaspar, and C. Nouyrigat Enzyme immunoassay for measurement of antibodies to herpes simplex virus infection: comparison with complement fixation, immunofluorescent-antibody, and neutralization techniques. J. Clin. Microbiol. 11: Dreesman, G. R., D. 0. Matson, R. J. Courtney, E. Adam, and J. L. Melnick Detection of herpesvirus type-specific antibody by a micro solid phase radioimmunometric assay. Intervirology 12: Eberle, R., and R. J. Courtney Assay of typespecific and type-common antibodies to herpes simplex virus types 1 and 2 in human sera. Infect. Immun. 31: Eisenberg, R. J., M. Ponce de Leon, L. Pereira, D. Long, and G. H. Cohen Purification of glycoprotein gd of herpes simplex virus types 1 and 2 by use of monoclonal antibody. J. Clin. Microbiol. 41: Forghani, B., N. J. Schmidt, and E. H. Lennette Solid-phase radioimmunoassay for typing herpes simplex viral antibodies in human sera. J. Clin. Microbiol. 2: GlIman, S. C., and J. J. Docherty Detection of antibodies specific for herpes simplex virus in human sera by the enzyme-linked immunosorbent assay. J. Infect. Dis. 136(Suppl): Nahmias, A., and W. Dowdle Antigenic and biologic differences in Herpesvirus hominis. Prog. Med. Virol. 10: Nahmias, A. J., I. Del Buono, J. Pipkln, R. Hutton, and C. Wickliffe Rapid identification and typing of herpes simplex viruses types 1 and 2 by a direct immunofluorescence technique. Appl. Microbiol. 22: Nahmias, A. J., W. R. Dowdle, J. L. Kramer, C. F. Luce, and S. C. Mansour Antibodies to Herpesvirus hominis types 1 and 2 in the rabbit. J. Immunol. 102: Nahmias, A. J., W. E. Josey, Z. M. Naib, C. F. Luce, and A. Duffey Antibodies to Herpesvirus hominis types 1 and 2 in humans. I. Patients with genital herpetic infections. Am. J. Epidemiol. 91: Patterson, W. R., W. E. Rawls, and K. 0. Smith Differentiation of serum antibodies to herpesvirus types 1 and 2 by radioimmunoassay. Proc. Soc. Exp. Biol. Med. 157: Pauls, R. P., and W. R. Dowdle A serologic study of Herpesvirus hominis strains by microneutralization tests. J. Immunol. 98:

5 VOL. 18, 1983 HSV TYPE-SPECIFIC ANTIBODIES BY ELISA Pereira, L., D. V. Dondero, D. GaLo, V. Devlan, and J. D. Woodle Serological analysis of herpes simplex viruses 1 and 2 with monoclonal antibodies. Infect. Immun. 35: Perefra, L., T. Klassen, and J. R. Barlnger Typecommon and type-specific monoclonal antibody to herpes simplex virus type 1. Infect. Immun. 29: Phummer, G A review of the identification and titration of antibodies to herpes simplex viruses type 1 and type 2 in human sera. Cancer Res. 33: Rawla, W. E., K. Iwamoto, E. Adam, and J. L. Melnkk Measurement of antibodies to herpesvirus types 1 and 2 in human sera. J. Immunol. 104: Schuewels, K. E., and A. J. Nahmla Antigens of herpes simplex virus type 1 and 2-immunodiffusion and inhibition passive hemagglutination studies. Z. Immunitaetsforsch. Immunobiol. 141: Seth, P., s. S. Prakah, and D. Ghosh Antibodies to herpes simplex virus types 1 and 2 in patients with squamous-cell carcinoma of the uterine cervix in India. Int. J. Cancer 22: Shore, S. L., C. M. Black, F. M. Melewkz, P. A. Wood, and A. J. Nahmlas Antibody-dependent cell-mediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus. J. Immunol. 116: Smith, J. W., E. Adam, J. L. Melnick, and W. E. Rawls Use of the 51Cr release test to demonstrate antibody responses to herpes simplex types 1 and 2. J. Immunol. 116: Smith, J. W., S. P. Lowry, J. L. Mehlick, and W. E. Rawls Antibodies to surface antigens of herpesvirus type 1 and type 2 infected cells among women with cervical cancer and control women. Infect. Immun. 5: Vestrgaard, B. F., and P. C. Grauballe ELISA for herpes simplex virus (HSV) type-specific antibodies in human sera using HSV type 1 and type 2 polyspecific antigens blocked with type-heterologous rabbit antibodies. Acta Pathol. Microbiol. Scand. Sect. B 87: Veseaad, B. F., P. C. Grauballe, and H. Spanggaard Titration of herpes simplex virus antibodies in human sera by the enzyme-linked immunosorbent assay (ELISA). Acta Pathol. Microbiol. Scand. Sect. B 85: Vofer, A., D. E. Bidwell, and A. Bartlett Microplate enzyme immunoassays for the immunodiagnosis of virus infections, p In N. R. Rose and H. Friedman (ed.), Manual of clinical immunology. American Society for Microbiology, Washington, D.C. Downloaded from on July 20, 2018 by guest