JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1979, p. 595-597 0095-1137/79/10-0595/03$02.00/0
Vol. 10, No. 4
Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Murine Hepatitis Virus ROBERT L. PETERS,* MICHAEL J. COLLINS, ANDREW J. O'BEIRNE, PAMELA A. HOWTON, SARA L. HOURIHAN, AND SARAH F. THOMAS Virus Diagnostic Laboratory, Microbiological Associates, Bethesda, Maryland 20016 Received for publication 16 July 1979
An enzyme-linked immunosorbent assay was developed for the detection of antibodies to murine hepatitis virus. A high prevalence of antibody to murine hepatitis virus was found by the enzyme-linked immunosorbent assay in colonies with a low prevalence of complement-fixing antibodies. Murine hepatitis virus strain A59 was found to be broadly reactive as an enzyme-linked immunosorbent assay antigen.
Antibodies to murine hepatitis virus (MHV) are most frequently measured by the complement fixation (CF) test (3, 7, 9). Titers determined by CF are usually low (1:10 to 1:40) in serum samples received in the Murine Virus Diagnostic Laboratory at Microbiological Associates. These sera are predominantly from retired breeder mice. In a previous study (7) involving two groups of mice 4 to 6 and 9 to 13 months of age, CF antibody was found in fewer than 50% of the older mice and infrequently in the group of mice 4 to 6 months of age. In a similar study (3), naturally infected breeder colony mice, 4 weeks or older, were found to transmit infectious virus. Detectable CF antibody increased with age from 15% positive at 4 weeks to 62 to 83% positive at 8 to 10 weeks. Antigenic relationships among the mouse hepatitis group of viruses have been demonstrated repeatedly by CF and the neutralization test (NT) (4-6). Recently, NT was found to be a sensitive diagnostic procedure for MHV antibodies (S. A. Stohlman, personal communication). In our study, a number of sera from naturally exposed mouse colonies were assayed by CF and NT. It was found that antibodies were most often detected by the A59 strain as opposed to the S or JHM strains of MHV. The enzymelinked immunosorbent assay (ELISA) was developed for the detection of MHV antibodies, using the A59 strain and later a polyvalent antigen (four strains) as the test antigen. We, therefore, further compared results of assays for MHV antibodies by CF, NT, and the ELISA on sera from natural and experimental infections of mice. A number of sera were also tested by the indirect fluorescent-antibody (IFA) procedure. The CF test used in this study was reported previously (8) and utilizes a polyvalent antigen consisting of MHV strains A59, S, 1, and JHM.
NT was conducted in Falcon Microtest II tissue culture plates (Falcon Plastics, Oxnard, Calif.). A single well was used per dilution, and known and unknown sera were diluted twofold from 1: 10 to 1:1,280 and 1:320, respectively, and mixed with an equal volume of virus containing 100 plaque-forming units per 0.05 ml. This mixture was incubated at 370C for 1 h, after which 5 x 104 NCTC 1469 cells were added in 0.05 ml of Eagle minimum essential medium with Earle balanced salt solution containing 20% fetal bovine serum (Microbiological Associates, Bethesda, Md.). Plates were read for cytopathic effect at 24 and 48 h, and the highest dilution without cytopathic effect was the neutralization titer of a given serum. Procedures used in ELISA were essentially those described by Voller et al. (10). Later testing was modified by shortening all incubation periods to 1 h at 37°C. Goat immunoglobulin G prepared against mouse immunoglobulin G (heavy chain) was supplied by Huntingdon Research Laboratories through the Resources and Logistics Branch of the Virus Cancer Program of the National Cancer Institute. MHV A59 antigen was prepared by harvesting cells and supernatant fluid from 18- to 24-h infected and control (noninfected) NCTC 1469 cells. This was frozen and thawed three times and clarified at 2,000 rpm for 20 min. Polyvalent antigen for ELISA was prepared by the same method as described for the CF test (8). Working dilutions for antigen and alkaline phosphatase-labeled anti-immunoglobulin G were determined by box titrations in polystyrene plates (Cooke Engineering Co., Alexandria, Va.). ELISA titers were calculated as the reciprocal of the highest dilution with an optical density value of :0.12 at 400 nm, based on 43 measurements against MHV A59 antigen of known negative sera on an ELISA reader (2). This value
595
596
NOTES
J. CLIN. MICROBIOL.
the result of a mean optical density of 0.04 imal reactivity of ELISA at day 7 was presum99% confidence interval of 0.07. For tests ably due to the fact that ELISA was heavy chain against MHV polyvalent antigen, results were specific, whereas the CF and IFA tests might read visually on a subjective scale of 0 to 4+. Of detect the immunoglobulin M class of antibodies 897 wells read visually and by the ELISA reader which would classically be present in the early preliminary to these studies, 481 were positive immune response. Sera from nine control mice, by the reader, and 461 (95.8%) were positive tested three at a time on days 0, 21, and 35, were visually; 445 were negative by the reader, and negative by all assays. 436 (97.9%) were negative visually. IFA tests In experiments shown in Table 2, sera were were also conducted on some sera, and results selected and grouped from several colonies based were read on a subjective scale of 0 to 4+. on CF titers against polyvalent antigen and then Positive and negative controls were included in tested in ELISA against the same polyvalent all assays. The IFA conjugate (Microbiological antigen. Sera having
Vol. 10, No. 4
Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Murine Hepatitis Virus ROBERT L. PETERS,* MICHAEL J. COLLINS, ANDREW J. O'BEIRNE, PAMELA A. HOWTON, SARA L. HOURIHAN, AND SARAH F. THOMAS Virus Diagnostic Laboratory, Microbiological Associates, Bethesda, Maryland 20016 Received for publication 16 July 1979
An enzyme-linked immunosorbent assay was developed for the detection of antibodies to murine hepatitis virus. A high prevalence of antibody to murine hepatitis virus was found by the enzyme-linked immunosorbent assay in colonies with a low prevalence of complement-fixing antibodies. Murine hepatitis virus strain A59 was found to be broadly reactive as an enzyme-linked immunosorbent assay antigen.
Antibodies to murine hepatitis virus (MHV) are most frequently measured by the complement fixation (CF) test (3, 7, 9). Titers determined by CF are usually low (1:10 to 1:40) in serum samples received in the Murine Virus Diagnostic Laboratory at Microbiological Associates. These sera are predominantly from retired breeder mice. In a previous study (7) involving two groups of mice 4 to 6 and 9 to 13 months of age, CF antibody was found in fewer than 50% of the older mice and infrequently in the group of mice 4 to 6 months of age. In a similar study (3), naturally infected breeder colony mice, 4 weeks or older, were found to transmit infectious virus. Detectable CF antibody increased with age from 15% positive at 4 weeks to 62 to 83% positive at 8 to 10 weeks. Antigenic relationships among the mouse hepatitis group of viruses have been demonstrated repeatedly by CF and the neutralization test (NT) (4-6). Recently, NT was found to be a sensitive diagnostic procedure for MHV antibodies (S. A. Stohlman, personal communication). In our study, a number of sera from naturally exposed mouse colonies were assayed by CF and NT. It was found that antibodies were most often detected by the A59 strain as opposed to the S or JHM strains of MHV. The enzymelinked immunosorbent assay (ELISA) was developed for the detection of MHV antibodies, using the A59 strain and later a polyvalent antigen (four strains) as the test antigen. We, therefore, further compared results of assays for MHV antibodies by CF, NT, and the ELISA on sera from natural and experimental infections of mice. A number of sera were also tested by the indirect fluorescent-antibody (IFA) procedure. The CF test used in this study was reported previously (8) and utilizes a polyvalent antigen consisting of MHV strains A59, S, 1, and JHM.
NT was conducted in Falcon Microtest II tissue culture plates (Falcon Plastics, Oxnard, Calif.). A single well was used per dilution, and known and unknown sera were diluted twofold from 1: 10 to 1:1,280 and 1:320, respectively, and mixed with an equal volume of virus containing 100 plaque-forming units per 0.05 ml. This mixture was incubated at 370C for 1 h, after which 5 x 104 NCTC 1469 cells were added in 0.05 ml of Eagle minimum essential medium with Earle balanced salt solution containing 20% fetal bovine serum (Microbiological Associates, Bethesda, Md.). Plates were read for cytopathic effect at 24 and 48 h, and the highest dilution without cytopathic effect was the neutralization titer of a given serum. Procedures used in ELISA were essentially those described by Voller et al. (10). Later testing was modified by shortening all incubation periods to 1 h at 37°C. Goat immunoglobulin G prepared against mouse immunoglobulin G (heavy chain) was supplied by Huntingdon Research Laboratories through the Resources and Logistics Branch of the Virus Cancer Program of the National Cancer Institute. MHV A59 antigen was prepared by harvesting cells and supernatant fluid from 18- to 24-h infected and control (noninfected) NCTC 1469 cells. This was frozen and thawed three times and clarified at 2,000 rpm for 20 min. Polyvalent antigen for ELISA was prepared by the same method as described for the CF test (8). Working dilutions for antigen and alkaline phosphatase-labeled anti-immunoglobulin G were determined by box titrations in polystyrene plates (Cooke Engineering Co., Alexandria, Va.). ELISA titers were calculated as the reciprocal of the highest dilution with an optical density value of :0.12 at 400 nm, based on 43 measurements against MHV A59 antigen of known negative sera on an ELISA reader (2). This value
595
596
NOTES
J. CLIN. MICROBIOL.
the result of a mean optical density of 0.04 imal reactivity of ELISA at day 7 was presum99% confidence interval of 0.07. For tests ably due to the fact that ELISA was heavy chain against MHV polyvalent antigen, results were specific, whereas the CF and IFA tests might read visually on a subjective scale of 0 to 4+. Of detect the immunoglobulin M class of antibodies 897 wells read visually and by the ELISA reader which would classically be present in the early preliminary to these studies, 481 were positive immune response. Sera from nine control mice, by the reader, and 461 (95.8%) were positive tested three at a time on days 0, 21, and 35, were visually; 445 were negative by the reader, and negative by all assays. 436 (97.9%) were negative visually. IFA tests In experiments shown in Table 2, sera were were also conducted on some sera, and results selected and grouped from several colonies based were read on a subjective scale of 0 to 4+. on CF titers against polyvalent antigen and then Positive and negative controls were included in tested in ELISA against the same polyvalent all assays. The IFA conjugate (Microbiological antigen. Sera having
