JOURNAL OF CLINICAL MICROBIOLOGY, May 1992, p. 1307-1311 0095-1137/92/051307-05$02.00/0 Copyright ©) 1992, American Society for Microbiology

Vol. 30, No. 5

Detection of Human Group C Rotaviruses by an Enzyme-Linked Immunosorbent Assay Using Monoclonal Antibodies RITSUSHI FUJII,l.2* MITSUTAKA KUZUYA,' MASAKO HAMANO,1 MASAO YAMADA,2 AND SHUDO YAMAZAKJ3 Department of Microbiology, Okayama Prefectural Institute for Environmental Science and Public Health, Okayama 701-02,1 De,partment of Virology, Okayama University Medical School, Okayama 700, and National Institute of Health, Tokyo 141,3 Japan Received 16 September 1991/Accepted 10 February 1992

An enzyme-linked immunosorbent assay using monoclonal antibodies was established for the detection of human group C rotaviruses. Seventeen clinical samples which were found to contain group C rotaviruses were all strongly positive, whereas 9 samples containing group A rotaviruses and 51 samples lacking rotaviruses were all negative with this test.





major causative agents of

acute viral gastroenteritis in infants and young children. The

ordinary rotaviruses have been classified as group A, and atypical rotaviruses that differ serologically from group A rotaviruses have been classified as groups B, C, D, and E (3, 21, 22). Epidemics caused by human group B or E rotaviruses have occurred mainly in China (6, 12, 13, 18, 28, 31). Incidences of human group C rotavirus infection have been increasing and have been reported almost every year at various locations in Japan (17, 20) and several other countries (1, 5, 8, 9, 10, 19, 23, 24). The in vitro cultivation of human group A rotaviruses has been achieved (30, 32), and a range of serological procedures employing antigen capture techniques, including the radioimmunoassay (7), enzyme-linked immunosorbent assay (ELISA) (29), latex agglutination (25), and reverse passive hemagglutination (26), are available for the detection of group A rotaviruses in clinical samples. Although the in vitro cultivation of human rotaviruses other than group A has not been successful yet, an ELISA specific for the human group B rotaviruses was established by using monoclonal antibodies (MAbs) as the capture antibody and hyperimmune serum as the detector antibody (4). On the other hand, the diagnostic procedures so far introduced for group C rotaviruses have some potential problems. Although serological identification by immunoelectron microscopy using hyperimmune reference antisera would be the best way to diagnose group C rotaviruses specifically, there are not enough reference antisera to monitor all the cases of viral gastroenteritis. An alternative method, polyacrylamide gel electrophoresis (PAGE) of viral RNA, is now used more frequently to identify group C rotaviruses. However, from a practical standpoint, it is difficult to use these methods in most diagnostic laboratories because both methods require special equipment or arrangements. Our study was undertaken to establish a rapid and simple diagnostic procedure specific for group C rotaviruses. A sandwich ELISA using MAbs enabled us to detect group C rotaviruses efficiently in fecal suspensions without any further purification. The difficulties involved in studying atypical rotaviruses including human group C rotaviruses are mainly related to *

the lack of a reliable in vitro culture system. We used a clinical specimen as a source of group C rotavirus for immunization and for the screening of MAbs. Because group C reference antisera were not available, we identified the presence of group C rotavirus in the sample by electron microscopy (EM) and RNA PAGE of the viral genome (11). The stool sample containing human group C rotavirus was homogenized and suspended in phosphate-buffered saline (PBS; pH 7.5) to make a 10% suspension. The fecal suspension was mixed with an equal volume of trichlorotrifluoro-





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FIG. 1. Polypeptide analysis of purified virions. Lane 1, clinical isolate used for immunization; lane 2, Wa strain of human group A rotavirus. The positions of molecular size markers (kilodaltons) are shown on the right. The major polypeptides of group C rotavirus are indicated by arrowheads.

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FIG. 2. Competitive binding curves in the antigen detection ELISA. The competitor MAbs were 5A12 (V), 12G6 (0), and 2H7 (reactive to human group A rotaviruses) (U). Also shown are results for no MAb (O), indicating results obtained with PBS instead of a competitor MAb.

ethane and centrifuged at 350 x g for 10 min. The aqueous phase was carefully removed, and the organic-aqueous interface was reextracted twice with PBS. The aqueous phase was pooled and overlaid on 40% sucrose. After centrifugation at 150,000 x g for 1 h at 4°C, the precipitate was resuspended in PBS. This preparation was referred to as crude human group C rotavirus. The crude human group C rotavirus was further purified by CsCl density gradient equilibrium centrifugation (starting refractive index, 1.38) at 100,000 x g for 18 h at 15°C. The banded material was collected and dialyzed against PBS. This viral preparation was referred to as a purified human group C rotavirus. The presence of double-shelled viral particles was checked by EM. Sodium dodecyl sulfate (SDS)-PAGE of polypeptides was carried out by using a 10% polyacrylamide slab gel (14, 15). The SDS-PAGE pattern of purified virion polypeptides (Fig. 1) was similar to that of group C rotaviruses in previous reports (2, 9, 14). Male BALB/c mice were immunized with the purified human group C rotavirus as previously described (4). Splenocytes were harvested and fused with murine myeloma XAg63 cells with polyethylene glycol 4000. The fusion mixture was plated in 96-well tissue culture plates with SFM-101 medium (Nissui Pharmaceutical, Tokyo, Japan). After 2 weeks, the surviving hybrids were screened for the production of antibodies against group C rotavirus with an ELISA. Positive cultures were expanded and subcloned twice by limiting dilutions. Ascites fluids were produced in pristane-primed BALB/c mice, and MAbs were purified from ascites fluids by Econo-Pac Protein A (Bio-Rad Laboratories, Richmond, Calif.). A MAb for the detector antibody was conjugated with N-biotinyl-w-aminocaproic acidN-hydroxysuccinimide ester (Enzo Diagnostics, New York, N.Y.). The isotypes of the MAbs were determined by a commercially available test kit (Serotec, Oxford, United Kingdom). The antigen detection ELISA using the MAbs was performed as follows. Microtiter plates were coated with 100 ,ul

of purified MAb (10 ,ug/ml in PBS) as the capture antigen overnight at 4°C. Each well was blocked with 2% dry milk (diluted in borate buffer; pH 8.0) for 30 min at room temperature. The 10% fecal suspension, which was prepared in borate buffer containing 2% dry milk and Tween 20 (BBS-T), was centrifuged at 350 x g for 10 min. Fifty microliters of the supernatant was added to the wells, and the plates were incubated overnight at 4°C. The plates were washed twice with PBS containing 0.05% Tween 20 (PBS-T), and 100 pl of biotin-conjugated MAb (0.5 ,ug/ml in BBS-T) 3.0

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Dilution FIG. 3. Sensitivity of the ELISA. Twofold dilutions of a 10% fecal suspension of a stool specimen were tested by ELISA. Samples exhibiting an OD450 greater than 0.130 were considered positive.

VOL. 30, 1992

was added to each well. Then the plates were kept for 1 h at room temperature. After three additional washes with PBS-T, 100 ,ul of horseradish peroxidase-conjugated streptavidin (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) was added to the wells and the plates were maintained for 10 min at room temperature. After a final four washes in PBS-T, 100 ,ul of the substrate was added to each well. The substrate solution consisted of 0.04% 3,3',5,5'-tetramethylbenzidine and 0.02% H202 diluted in citrate phosphatebuffered saline (pH 5.0). After 10 min of incubation, the yellow color produced by adding 100 ,ul of 1 M phosphoric acid was measured by an Immunoreader NJ-2000 (Nunc, Roskilde, Denmark) at a wavelength of 450 nm. Two MAbs, 5A12 and 12G6, were obtained by screening hybridomas produced after the immunization of mice with the purified group C rotavirus. The specificity of these MAbs to group C rotaviruses was first checked by an ELISA by using crude antigens and then confirmed by immunoelectron microscopy by using the purified human group C rotavirus (data not shown). The isotype of these MAbs was found to be IgG2b. To determine whether the MAbs reacted with the outer or inner capsid, the purified human group C rotavirus was treated with various concentrations of EDTA (2, 5, and 10 mM) for 15 min at room temperature. The treated preparation was then tested for reactivity in the ELISA by using the homologous MAbs as the capture antibody and the detector antibody. The reactivities with MAbs 5A12 and 12G6 were both drastically reduced by the treatment of the virus with EDTA at concentrations of 2 mM and higher. For further epitope mapping, the competitive binding assay described by Bums et al. (4) was employed with minor modifications. The purified human group C rotavirus reacted with various concentrations of each competitor MAb overnight at 4°C. The virus-MAb complexes were introduced to the wells coated with the purified MAb 5A12. The rest of the ELISA was performed by using 5A12 as the detector MAb. MAb 12G6 partially inhibited MAb 5A12 (Fig. 2). These findings indicated that the two MAbs, 5A12 and 12G6, recognize overlapping (but not identical) epitopes on the outer capsid of rotaviruses. Western blot (immunoblot) analysis to identify the molecular weight of the antigen recognized by these MAbs was unsuccessful, suggesting that these MAbs seem to recognize conformation-dependent

epitopes (16, 27). To optimize the ELISA system for the specific and sensitive detection of group C rotaviruses, combinations of a capture antibody and a detector antibody were examined. Because the best result was obtained with the protocol using 5A12 as the capture antibody and 12G6 as the detector antibody, this combination was used thereafter.

Five clinical isolates which had been identified as group C rotaviruses by other investigators using reference antisera (17, 20) were used to check the specificity of the ELISA. Three of these isolates were kindly given to us by the Ehime Prefectural Institute, while the other two were given by the Fukui Prefectural Institute. The Wa strain of human group A rotavirus was propagated in MA104 cells with medium containing 4 ,ug of trypsin per ml (30). Five clinical isolates which were found to contain group C rotaviruses by reference antisera were all strongly positive in the ELISA, whereas the standard Wa strain of group A rotavirus was negative for this test (see Fig. 4). To determine the sensitivity of the ELISA, twofold dilutions of the 10% stool suspension containing a group C rotavirus were tested. The amount of rotavirus in the sample


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Columns FIG. 4. Reactivities with the antigen detection ELISA. Column of group C rotaviruses identified with reference 1, five samples antisera. RNA profiles of all these samples were pattern I. Column 2, 17 clinical samples which were found to contain group C rotaviruses by RNA PAGE. RNA profiles of 11 samples (A) were pattern I, and RNA profiles of 6 samples (A) were pattern II. Column 3, group A samples (*, Wa strain; O, nine clinical samples which were found to contain group A rotaviruses by RNA PAGE). Column 4, 51 clinical samples (0) in which no rotavirus was detectable. Samples exhibiting values greater than 0.130 were considered positive.


determined by EM as described previously (25). The end point of the positive reaction in the ELISA was the 1:25 dilution (Fig. 3). The end point sample was estimated to contain approximately 3.28 x 106 viral particles per ml. To evaluate the practical usefulness of the ELISA system, 77 stool samples were obtained from patients with acute diarrhea during epidemics of acute gastroenteritis that occurred in Okayama from 1989 to 1990. Ten percent suspensions of these stool samples were tested with the ELISA (Fig. 4). Seventeen samples which had been determined to contain group C rotaviruses by RNA PAGE were all strongly The patterns of RNA PAGE shown positive for the ELISA. into two groups (Fig. 5). One divided were isolates these by pattern I was pattern (Fig. 5, lane 3) tentatively designated identical to that of the five isolates identified by reference antisera, while the other (pattern II; lane 4) was similar but not identical to them. Segment 10 in pattern II characteristhat of pattern I. No tically migrated more slowlyofthan the ELISA was observed difference in the reactivity between the two patterns. On the other hand, 9 samples containing group A rotaviruses and 51 samples from which





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Nevertheless, it is likely that more specimens over a period of time and additional geographical locations should be examined before this ELISA system can be evaluated conclusively. In conclusion, we have established a sandwich ELISA system using MAbs for the specific detection of group C rotaviruses directly from clinical specimens. This procedure can be widely used not only for the monitoring of outbreaks of viral gastroenteritis in epidemiological studies but also for the rapid diagnosis of individual patients at hospitals. terns.

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We thank M. Mori, Ehime Institute of Public Health, and K. Matsumoto, Fukui Prefectural Institute of Public Health, for providing clinical isolates and S. Kawano, Department of Pediatrics, .-














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Detection of human group C rotaviruses by an enzyme-linked immunosorbent assay using monoclonal antibodies.

An enzyme-linked immunosorbent assay using monoclonal antibodies was established for the detection of human group C rotaviruses. Seventeen clinical sa...
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