| The Chicken stunt and immunosuppressive syndrome virus (CSISV) is mul tiplied by inoculating to the allantoic cavity of SPF chicken embryo. Then the allantoic fluid is collected and the virus is purified by differential centrifugati on. Using the purified virus immunized Balb/c female mice. The splenic cell o f immunized mice and NSO myeloma cell are fused under the induction of PE G-1500. The fusion ratio is 96.88% and the positive ratio is 4.84%. A cell str ain which can excrete antibody to CSISV and the cell strain is harvested and i s named 3H10 by indirect ELISA and limited dilution cloning. The tite of 3H 10 cell supernatant is 1:320 and The rats ascites which was made by 3H10 ce 11 is 1:8.2×106 by index-ELISA. The hybridoma cell can excrete antibody(3 H10-McAb) stably by serial passage,freezing and resuscitation.The subgroup of 3H10-McAb is IgG2a by capture ELISA. Index-ELISA is used to detect the specificity. The result showed the 3H10-McAb has no reaction with negative allantoic fluid; It also has no cross reaction with IBDV,NDV,IBV. It means the 3H10-McAb has good specificity. Western-blot convinced it can react specifically with the protein p30 of the CSISV.Using the McAb optimized coditions of immunochemistry. The best conditions are:ascites is diluted with 1:80 and works for 4℃overnight; HRP-IgG is diluted with 1:30 and works for 37℃/30min; DAB works for 5 min, Harry's hematoxcylin is diluted with 1:5 and works for 25s.Using the optimized conditions estabilished the HRP-immunohistochemistry and detected the the kidney and liver of broiler chicken which were inoculated CSISV artificially.The results are all positive. It means the 3H10-McAb can bind with the virus in tissues very well and can detect the CSISV in infected tissues effectively.The successful preparation of the McAb against CSISV lays foundation for establishing the immunologic diagnostic method of the disease. It also lays a good foundation for the futher research of the disease. |
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