Preparation Of McAb Against Dairy Cow L-selectin And Study Of Express Disciplinarian On Neutrophil Of Mastitis Dairy Cow

Staphylococcus aureus is the main pathogen that causes mastitis. It is a troublesome problem that staphylococcus aureus causes mastitis in clinical veterinary medicine. Immunity therapeutics provides a new pathway undoubtedly for solving the problem. The therapy of bispecific antibodies have been one of these hot-spots, because bispecific antibodies possesses better prospect of clinical application that can kill Staphylococcus aureus. Bispecific antibodies possesses two various antigen binding sites, which can connect target pathogens with immunologic cells. Bootstrapped immunologic cells kill target pathogens.In the experiment, Neutrophil was separatied from peripheral blood of dairy cow by Percoll gradient centrifugation, purity>95%, viability>97%, concentration is 6×107cells/ml. The BALB/C mice were inoculated intraperitoneally with 0.5ml neutrophils. The booster inoculations with the same donor cow were performed similarly in each mouse at 2-week intervals.NO.1,2,3 of first team mice were detected titer of blood after the third inoculation, the mouse which antibody titer was the highest was selected for spleen collection. The second team mice for four inoculations. L-selectin was used as antigen to screen positive holes by indirect ELISA. Hybrid tumor cells were cloned by means of limiting dilution to created hybridoma cell strain, namely 1F6, 3C2 and 4H3. Modal number of 3 strain hybridoma are 82～95,88～95 and 86～94 respectively;The titer of supernatant of 3 strain hybridoma are 1:400, 1:600 and 1:800 respectively. The titer of ascitic fluid of 1F6, 3C2 and 4H3 were 1:6×104, 1:8×105 and 1:1.3×105 respectively . Ig subset were IgG1,IgG2a,IgG1 respectively. The best coating concentrations of antigen conjugating the antibodies were 0.0625μg/ml,0.125μg/ml and 0.500μg/ml respectively, the best dilution were 1:800, 1:3200 and 1:6400 respectively, and affinity constants were 3.74×107,1.6×109 and 2.42×109 M-1 respectively. The monoclonal antibodies relative affinity is 4H3>3C2>1F6,and different antigen binding epitope, and they could specifically bind with the 95.0kDa of PMN membrane protein but not other membrane proteins, I.e. anti-CD62L McAb.The express disciplinarian of L-Selectin on PMN that came from healthy dairy cattle and clinical mastitis dairy cattle was detected, Using flow cytometer and low voltage scanning electron microscope by indirect immunofluorescence technique and Immunogold staining technique.Result indicated: The intensity of L-selectin that PMN expresses in clinical mastitis is significant.The result provid theory for our study to prepare a new type drug of treat dairy cattle mastitis.