An improved enzyme-linked immunosorbent assay for the detection of rotavirus in faeces of neonates

Abstract

A direct enzyme-linked immunosorbent assay (EIA) for the detection of rotavirus in neonatal stools was developed. Rabbit antiserum against SA 11 rotavirus was incorporated as both coating and detector antibody, and rotavirus-negative rabbit serum was applied as a coating antibody control to eliminate false positive results. Pretreatment of stools with EDTA was found to increase both the sensitivity and specificity of the assay. This effect was greatest when 0.25 M EDTA (tetrasodium salt) was included in homogenized stool suspensions before the removal of solid debris by centrifugation. By electron microscopy, this EDTA pretreatment appeared to partly uncoat human rotavirus particles in faeces. Potentially suitable solid phase supports and horseradish peroxidase substrates were evaluated in the development of the assay. Screening of stool samples revealed that repeated freezing and thawing of stools eliminated positive EIA reactions. The SA 11 coating antibody compared favourably with a reference coating antiserum prepared against human faecal rotavirus strains. This EIA showed greater sensitivity for rotavirus detection than electron microscopy of stool concentrates prepared by ultracentrifugation, on testing 143 stools from 99 neonates and children. The assay has been applied successfully to detection of rotavirus in stools of neonates containing meconium, smaller amounts of viral antigen than in older children, and lacteal antirotaviral antibody. It is likely to be particularly useful for cross-infection studies in hospital wards and neonatal nurseries.