The Development And Identification Of The Monoclonal Antibody Against Luteinizing Hormone

To estabolish the quick test for LH in the sera, LH was coupled to carrier protein of bovine serum albumin (BSA) using carbodiimide method. The LH was used as immunogen and coating antigen, then BALB/c mice were immunised with LH-BSA and LH. The titer of anti-LH serum was tested with indirectly enzyme linked immnosorbent assay (ELISA). The optimised working concentrations of coated antigen and antibody were performed with phalanx titration.The constantly secreting anti-LH monoclonal antibody (McAb) was screened with cell fusion and hybridomas techinque. The potency of McAbs was determined with indirect ELISA. The affinity constant and the cross-reaction rate of LH-McAbs were measured by competitive ELISA. The result indicated that:1 The indirect ELISA method, coated antigen 1.0μg/mL LH dissolved in carbonate buffer solution (CBS, pH9.6, 0.05 mol/L),with HRP-labelled goat anti mouse IgG (anti Ms IgG (H+L) /HRP) diluted by PBS (pH7.4, 0.1 mol/L) in l∶1 500 as a secondary antibody, 1.5% gelatinum as a blocking agent, and TMB (3,3',5,5'-tetramethyl benzidine dihydrochloride)as a substrate, was developed to detect antibody against LH. The method's reproducibility was good and its coefficient of variability (CV) was 0.72%~ 1.07%. This manifested the method presented good results.2 The efficiency of confluent cells with 50% PEG (Mr 4 000) was 81%, and the rate of positive clone was 14.7%. Throuth 4 times screened, three hybridoma cell strains producing anti-LH monoclonal antibodies (McAb) were gained and named E11, B5 and D7, respectively.3 Intraperitoneal injections were to three BALB/c mice with three hybridoma cell strains, respectively. We obtain ascites from three mice.The characteristic of McAb was performed by ELISA, the titer of ascites was 1:1×105, the affinity constant of LH-McAb for coated complete antigen was 108. The cross-reaction rate of McAb to FSH and hCG was all below 0.5%.