Obtaining immunologically active bovine viral diarrhea virus (BVDV) proteins for enzyme linked immunosorbent assay (ELISA).
We report here the preparation of BVDV antigen (Ag) to obtain adequate quantities of pure active viral proteins from Madin Darby Bovine kidney (MDBK) cell culture infected with BVDV cytophatic Oregon C24V strain. SDS-PAGE, Immunodot, ECL-Western blot and ELISA showed the best specificity and activity of BVDV Ag obtained from infected cells only by mild experimental conditions. BVDV Ag preparations showed the peculiar BVDV sensitivity to proteases, nonionic surfactants and tend for protein degradation and irreversible loss of conformational antigenic determinants with subsequent inability to detect antibodies against viral Ag in hyperimmune sera. The widest panel of immunologically active and specific polypeptides, that elicit Ab production in hyperimmune sera, was obtained by ultrasonication and subsequent purification on 20%-50% sucrose cushion. We observed that BVDV tend to remain in the infected cells, to associate with components of serum and cellular origin--this is of crucial importance towards the specificity of ELISA. BVDV antigenic properties are determined by the labile conformational antigenic epitopes.
Pilinkiene
A
A
Institute of Immunology, Lithuania, Vilnius.
Pieskus
J
J
Salomskas
A
A
D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis*; D000956:Antigens, Viral / Q000276:immunology; D015153:Blotting, Western; D002417:Cattle; D001908:Diarrhea Viruses, Bovine Viral / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D012680:Sensitivity and Specificity
D000914:Antibodies, Viral; D000956:Antigens, Viral