Establishment Of Detection Methods For Hog Cholera Virus With SPA-COA And ELISA

CSFV-F₁₁₄ strain and HCLV strain were grown in PK-15 cell line and calf testicle cell line respectively. HCLV strain was harvested and concentrated against polyethylene glycol 6000 overnight by freezing and thawing. The hyperimmune serum of rabbit anti-HCLV and pig anti-HCLV were prepared by purified HCLV immunized rabbit and pig. Mainly immunoglobulin G (IgG) of rabbit and pig anti-HCLV were obtained by ammonium sulphate precipitation and subsequently unsalted with dialysis.The method of SPA co-agglutination was conceived for diagnosing CSFV from other viruses popularly, maneuverablely and intoitionisticly. We established the SPA plate co-agglutination test by SPA combining rabbit anti-HCLV hyperimmune serum and pig anti-HCLV hyperimmune serum respectively. Gradient dilutions of hyperimmune serum were coupled with SPA to prepare SPA diagnostic agent. It was identified that the optimal ratio of rabbit anti-HCLV hyperimmune serum was 1:1, and that of pig anti-HCLV hyperimmune serum was 1:2. The sensitivities of rabbit anti-HCLV SPA diagnostic agent and pig anti-HCLV SPA diagnostic agent were 1:32, 1:64 respectively by the comparison of two SPA combine hyperimmune sera. This method was more sensitive than Counter Immunoelectrophoresis which just detected 1:2 dilution Ag. The lymph node and spleen were the best detection parts by detecting different parts of clinical morbid animals. Furthermore, this method is handy, fast, economic and no special apparatus needed, which fit for fast identification of CSFV in common field and epidemiological investigation.The immunology method of indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established by using these three kinds of IgG The optimal dilutions of rabbit anti-HCLV, pig anti-HCLV and HRP-sheep anti-rabbit were 1:40,1:200,1:400 respectively. This method had high sensitivity, specificity and duplication, which fit for definitive diagnosis of CSFV in laboratory by the sensitivity test, the specific blocking test, cross and duplication test.50 clinical specimens were detected by SPA co-agglutination and indirect double antibody sandwich ELISA. 6 samples showed CSFV positive by SPA co-agglutination, and the positive rate was 12%, the detection results could be reported in 4⁷min. 10 samples showed CSFV positive by indirect double antibody sandwich ELISA, and the positive rate was 20%, it could be completed in 24h.