VP4 and VP7 typing using monoclonal antibodies

Abstract

Both rotavirus outer capsid proteins, VP4 and VP7, elicit neutralizing antibodies. Neutralizing mouse monoclonal antibodies (N-MAbs) to VP7 are easily derived and have been used widely and successfully to serotype both stool-derived and culture-adapted rotaviruses by enzyme immunoassay (EIA). Generally, approximately 70% of rotaviruses in stool samples are typable by VP7 EIA, an inexpensive and practical method. Variations in antigenic regions between strains within human rotavirus serotypes 1, 2, 4, and 9 have been recorded. These have been termed monotypes because they are detected with N-MAbs. The molecular basis for monotypes has been determined by mapping mutations selected in N-MAb-resistant antigenic variants, and by sequence analysis of the gene encoding VP7 in newly recognized monotypes. Antigenic regions A, B and C in VP7 are involved. In order to detect all members of a particular VP7 serotype, it is necessary to type with a panel of N-MAbs specific for that serotype. N-MAbs to VP4 of human rotavirus are difficult to raise and few have proven suitable for VP4 serotyping by EIA. The specificity of the assay for each P type is highest when the VP7 serotype specificity of the capture antiserum is matched to the G type of the rotavirus in the test sample. The VP4 EIA gives similar typing rates to the VP7 typing EIA. N-MAbs directed to VP8, the smaller subunit of VP4 generated by proteolytic cleavage, are more likely to show serotype specificity. Some N-MAbs that select mutations in the putative fusion region of VP5, the larger subunit of VP4, show cross-reactivity with extracts of normal, uninfected MA 104 cells and with fetal bovine serum. These N-MAbs also give elevated EIA OD readings with rotavirus-positive, but previously non-reactive fecal samples which have been frozen and thawed repeatedly. Overall, VP8-reactive N-MAbs appear most suitable for VP4 typing by EIA.