Simple and specific enzyme immunoassay using monoclonal antibodies for serotyping human rotaviruses

Abstract

An enzyme immunoassay for serotyping human rotaviruses in stools and in cell culture was developed. Hyperimmune rabbit antisera to rotaviruses were used as capture antibodies, and rotavirus-neutralizing mouse monoclonal antibodies specific for serotypes 1, 2, 3, and 4 were used as detection reagents. Partial purification of monoclonal antibodies and inclusion of skim milk powder in antibody diluents contributed to assay specificity. The sensitivity of this assay was greater than that of a direct enzyme immunoassay in which rotaviruses of the appropriate serotype were adsorbed directly to the solid phase. When fecal extracts were concentrated threefold, this serotyping enzyme immunoassay was of equal specificity and approached the sensitivity of electron microscopy for rotavirus detection. This assay is simple and rapid and is suitable for serotyping the large numbers of isolates obtained from epidemiological studies and vaccine trials. again in 7 days with virus in saline. Serum was collected 14 5(9

Rotavirus Antagonizes Cellular Antiviral Responses by Inhibiting the Nuclear Accumulation of STAT1, STAT2, and NF-κB

“…Cells were infected with RRV, Wa, SA11, or UK rotavirus for 6 h, left untreated or treated with IFN-␣, IFN-␥, or TNF-␣ for 30 min, and fixed with 3.7% (wt/vol) formaldehyde (Sigma) in phosphate-buffered saline (PBS) for 10 min, followed by permeabilization with acetone-methanol (1:1, vol/vol) at Ϫ20°C for 15 min. Fixed cells were blocked in 3% (wt/vol) bovine serum albumin (Sigma) in PBS for 30 min and reacted at 20°C for 1 h with a combination of polyclonal rabbit antibody (2 g/ml; Santa Cruz) to STAT1 (SC-345), STAT2 (SC-476), or p65 (SC-109) and mouse monoclonal antibody RVA to rotavirus VP6 (14). Cells washed with PBS were incubated for 30 min with a combination of Alexa Fluor 488-conjugated anti-mouse immunoglobulin G and Alexa Fluor 594-conjugated anti-rabbit immunoglobulin G (10 g/ml each; Invitrogen, Carlsbad, CA).…”

Nosocomial outbreak of neonatal gastroenteritis caused by a new serotype 4, subtype 4B human rotavirus

“…The different serotypes have been identified on the basis of their reactivity with animal hyperimmune sera in neutralization assays. The neutralization antigens responsible for serotype specificity correspond to two outer capsid proteins: VP7, the major neutralization antigen, which is the gene 8 or 9 product, and VP4, the minor neutralization antigen, coded for by the viral gene 4 [Greenberg et al, 1983b;Hoshino et al, 1985;Liu et al, 19881. Recently, several enzyme-linked immunosorbent assay (ELISA) systems have been developed for typing of strains directly in stools by using neutralizing monoclonal antibodies (MAbs) directed to VP7 of different HRV serotypes [Coulson et al, 1987;Taniguchi et al, 19871. In addition, two distinct subtypes of serotype 4 HRV strains, referred to as subtypes…”

Typing of human group A rotavirus with alkaline phosphatase‐labeled oligonucleotide probes or a monoclonal enzyme immunoassay in unfrozen stools of children with diarrhea in Bangkok

“…Rotavirus is an important cause of diarrhea worldwide [Kapikian and Chanock, 19851. An effective rotavirus vaccine must contain strains antigenically simi-nucleotide sequences coding for VP7 [Coulson et al, 1987;Green et al, 19881. The evaluation of rotavirus vaccines in clinical trials would benefit from a simple and accurate method to identify rotavirus G types. The technique should be accurate and suitable for use in laboratories in developing countries.…”