Preparation And Identification Of Monoclonal Antibody Against Bursin

Bursin(BS) was a bioactive compound,which was extracted from the BF and its molecular weight was 0.354kD.Not only did bursin induced the function of the B lymphocyte differentiation,but also promoted the generation of antibody and the immunized effectivness of vaccine,sustained the normal development of BF,prevented some viral infection and resisted to immunosuppression factor.Our experiment prepared the complete antigen of bursin with sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate (Sulfo-SMCC),produced monoclonal antibody against the BS and established the competing ELISA system of McAb against BS.BS was coupled with carrier protein bovin serum album(BSA) and prepared the immunizing antigen that immunized 8 week's female Balb/c mice.It also could be conjugated with carrier protein egg albumin(OVA) and produced the detection antigen which can detect the valency of mice blood serum and blot positive hybridoma cell.The immunized splenocyte of mice were fused with NS0 myeloma cells using PEG.One hybridoma cell line of BS-3F1 was obtained.The hybridoma cells was cloned by limited dilution method for three times, whose titers of cultivate medium was 1:100.The isotype of McAb was IgM.The result indicated that there was a transparent substance peak in 3.863 min by high performance liquid chromatography(HPLC). However,this peak was not presenced in the figure of exaction of BF and 3F1-McAb.The follow of immunohistochemistry indicated that the neuropile of BF occurred positive reaction.It certificated that BS existed in the BF can reaced with 3F1-McAb.Competitive inhibition ELISA also manifested that synthesized artificially BS can suppressed the reaction between the 3F1-MeAb and detected antigenImmunoassay of BS had been established with anti-BS monoclonal antibody 3F1- McAb by indirect ELISA.Our experiment also optimized the coating factor,the better kind of confining liquid,the response time and concentration of mouse-anti-goat antibody and substrate response time.The supernatant of 3F1 hybridoma cell was established to the system of indirect competing ELISA and builded the standard curve. The regression equation was y=-18.395x+37.937 and regression coefficient was 0.9704.The preparation of monoclonal antibody against BS provided the foundation for the immunologic mechanism and the determination of BS contents of chicken.