| African swine fever (ASF) is a highly lethal viral hemorrhagic fever of domestic pigs. Both mortality and mobility of the virulent strain infection can be100% with severe symptoms such as hemorrhage, breath difficulty and nervous disorder. Due to its difficulty for control and heavy loss, the disease is listed as one of important class A diseases in animals by OIE and Chinese Ministry of Agriculture. Although China is free of the disease presently, it remains a big threat for Chinese pig industry with the increasing international trade and frequent people exchange. Therefore, it is urgent to establish quick accurate diagnostic methods for preventing the disease.The VP72 is the conserved major coat protein of ASFV and is often used as the target antigen for diagnostic of the disease. The monoclonal antibody is high specific, can be persistently, infinitely supplied, now has been widely applied in the virus infected diagnose.To prepare the specific monoclonal antibodies against ASFV, a GST-VP72 fusion protein expressed in recombinant E.coli was used to immunize BALB/c mice, and the splenetic B cells were fused with SP2/0 cells. Five hybridoma cell lines stably secreting VP72-specific monoclonal antibodies were screened by using indirect ELISA against another E.coli-expressed fusion protein GST-VP72S. Subclass identification showed that the five monoclonal antibodies belonged to IgG2b.To establish an indirect ELISA for detection of ASFV antibodies, E.coli-expressed soluble fusion protein GST-VP72S was used as the coating antigen and the reaction conditions were optimized as follows: ?ug/ml of the coating antigen GST-VP72 in buffer bicarbonate (pH9.6), incubation for 2 hours at 37℃, blocking overnight with PBS containing 5% skimmed milk at 4℃, washing with PBS containing 0.5M NaCl and 0.5% Tween 20, and 800-fold dilution of the mimic pig antiserum containing ?-fold diluted ascetic monoclonal antibody.
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