Preparation and characterization of antibodies against reassortant rotavirus strain RV441

Abstract

Rotaviruses have been shown to be the major cause of infantile gastroenteritis in developed and developing countries. In developing countries, the serological assays are the main use of rotavirus detection but the commercially available reagents used for the assays are too expensive. The reference rotavirus antigens and antibodies representative of subgroups and serotypes are not yet available. A reassorthant rotavirus strain RV441 with subgroup I serotype 2 antigenic determinants was propagated in MA104 cells, precipitated with polyethyleneglycol, extracted with fluorocarbon and then used to immunize rabbit. Hyperimmune reaponses of rabbit was detected by ELISA. Immunoblotting analysis of rabbit antiserum showed that immune response directed strongly to VP2 and VP5 and weakly to VP6 and Vp7 structural proteins of rotavirus. Purified immunoglobulin was prepared form rabbit antiserum and used at the optimal dilutions to develop a double-sandwich ELISA to monitor the persence of rotavirus in clinical spectimens. A total of 260 fecal specimens from diarrheic patients were tested. Only 88 specimens showed high OD(,490) reading value (1.12 - 2.5), and all of them were short RNA electropherotypes. Other 12 specimens reacted weakly in the ELISA test (OD (,490) = 0.23 - 1.00), and had been confirmed possitive by blobking ELISA test. The remaining 160 specimens, composed of 100 specimens with long electropherotype and 60 specimens negative for rotavirus, all showed negative results in this ELISA. We proposed that this ELISA system may be of value in identifying subgroup I human rotavirus but further test of additional clinical specimens and other reference rotavirus strains are required to confirm the sensitivity and specificity of the method.