Radioimmunoassay for detection of the snow mountain agent of viral gastroenteritis

Abstract

The Snow Mountain Agent (SMA) is a Norwalk-like viral agent of acute gastroenteritis that has been detected only by immune electron microscopy (IEM). We established a solid phase microtiter radioimmunoassay (RIA) for SMA antigen employing pre-and post-challenge sera from volunteer studies as capture antibodies. SMA was detected in 12 of 67 stool samples from volunteers who were ill after oral challenge with SMA. All samples in which virus particles were detected by IEM were positive by RIA, but the RIA was 10-80 times more sensitive than IEM. To detect serum antibody to SMA, a blocking test was developed, employing diarrheal stool containing SMA as a standard source of antigen. Serum antibody rises were detected in eight out of nine volunteers with experimentally induced illness following challenge with SMA, as well as in three out of three naturally occurring cases. A preliminary sero-epidemiologic survey suggested that infection with SMA was common in the population surveyed. This RIA should permit large scale seroepidemiologic studies of SMA to be carried out, and should also facilitate characterization of this agent.

Production of a monoclonal antibody against the Snow Mountain agent of gastroenteritis by in vitro immunization of murine spleen cells.

“…SMA used these in experiments was derived from experimentally infected volunteers, all of whom were ill and subsequently seroconverted to SMA by radioimmunoassay or EIA (3,7,8 (19) containing 40% (vol/vol) fetal calf serum was added, and the culture was continued for a total of 3-5 days. On days 3, 4, and 5, an aliquot of spleen cells was removed and fused with nonsecreting X63-Ag8.653 myeloma cells at a 5:1 spleen/myeloma cell ratio by using 40% polyethylene glycol (Mr 1000; Aldrich) and 5% dimethyl sulfoxide in DMEM.…”

“…The specificity of SM-4 for SMA was further investigated by blocking EIA (8). Blocking assays were performed with human convalescent sera as the capture antibody, followed sequentially by antigen, the unlabeled antibody being tested, and finally by biotin-labeled second antibody, either human or monoclonal.…”

“…Several immunologically distinct agents have been described including the Norwalk agent (1), the Hawaii agent (2), and the Snow Mountain agent (SMA) (3). Radioimmunoassays and enzyme immunoassays (EIA) have been developed for the Norwalk agent (4-7), the SMA (7,8), and the Hawaii agent (unpublished data). The reagents used in these assays consist of sera and stool specimens from human volunteers or chimpanzees experimentally challenged with these viruses.…”

Diagnosis of viral gastroenteritis: limits and potential of currently available procedures

“…Immunoglobulin G (IgG) purified and radiolabeled with gamma-radioactive isotopes of iodine, such as 125-I, is used as a detector system. Although the RIA technique is extremely specific and 10 to 100 times more sensitive than IEM, it requires special precautions since radioactive substances are used [60][61][62]. RIA assays have been modified to detect virusspecific antibodies using a blocking format.…”

“…RIA assays have been modified to detect virusspecific antibodies using a blocking format. Several RIA blocking techniques have been developed for the detection of antibodies to noroviruses; these assays appeared to be 10 to > 200 times more sensitive than the IAHA [60][61][62].…”