Preparation and Identification of Murine Monoclonal Antibody against TNF-α

Objective To prepare and identify the murine monoclonal antibody(McAb)against tumor necrosis factor-α(TNF-α)with high neutralizing activity.Methods BALB/c mice were immunized with recombinant TNF-α,based on which the McAb was prepared by hybridoma technique,then purified and determined for titer by indirect ELISA and MTT method,for antigenic epi-topes by additive ELISA,for relative affinity index by ammonium thiocyanate elution method,and analyzed for class,subclass,speci-ficity and stability.The cell strains secreting McAbs were analyzed for chromosome karyotype.Results Seven cell strains secreting McAb against TNF-αwere screened.The McAbs secreted by 6 of the 7 strains showed high neutralizing activity and stability.All the McAbs secreted by 7 strains were IgG1 withκchain,and their neutralizing titers were 1:40～1:160 and(1～3)×10-4 in supernatant and ascites respectively.The chromosome karyotypes of the 7 cell strains were consistent with those of hybridoma cells.The antigenic epitope of McAb secreted by 5H10 strain was different from those by the other 6 strains.The relative affinity indexes of McAbs se-creted by the 7 cell strains were between 0.4 and 1.5 mol/L.Conclusion The murine McAb against TNF-αwith high neutralizing activity was successfully prepared,which laid a foundation of construction of chimeric antibody against TNF-α.