Preparation Of The Monoclonal Antibodies Against MPA And Establishment Of ELISA Detecting Method

Medroxyprogesterone acetate, MPA, an artificial progestogen, was used as a feed additive to promote the growth of animals and increase the feed conversion efficiency. It was banned for the increased risk of breast cancer in China and Europe. Many chemical and physical methods for detecting medroxyprogesterone acetate have been established by the international community. Though some of these methods are sensitive and accurate, they are not suitable for screening a large number of samples because these methods are time-consumption, and costly and highly skilled. Enzyme-linked immunosorbent assay (ELISA) as a rapid, special and sensitive biological method is gradually applied in drug residues assay. This research was designed to synthesize the antigen and developed the antibodies, and established the ELISA method for detecting MPA.Carboxymethyloxy method was adopted to synthesize MPA derivative. Then 3-carboxymethyloxyimine medroxyprogesterone acetate was conjugated with carrier proteins BSA and OVA by dicyclohexylcarbodiimide (DCC) for animal immunization and ELISA tests. The conjugates were confirmed by UV spectrophotometer and SDA-PAGE. Their molar ratios of the hapten and the carrier protein were 19:1 and 18:1.After BALB/c mice was immunized by MPS-BSA for 4 times, which was measured by indirect ELISA. The spleen cells of the immunized mice with the best immune effects were taken out and fused with SP2/0 cell in PEG..Two hybridoma cell lines producing monoclonal antibody (McAb) against medroxyprogesterone acetate ( MPA) were established by the hybridoma technique, which was designated as 4C11A3B6 and 4C11G8H6. The indirect ELISA titers of cell supernatant and ascites were 1:640 and 1:320, 2.56×106 and 1.6×106, respectively. The number of chromosome of the hybridoma cell lines was 99.64±1.5 and 99.35±1.5. McAb were of subclasses IgG2а.The competitive inhibition enzyme-linked immunosorbent assay (ciELISA) for MPA was established. The regression equation of this curve is y=-30.022LogC+90.191,R2=0.9911.the curve has a favorable linearity relation within the concentration range of 1～100 nag/ml. The IC50 value was 22ng/mL by competitive inhibition enzyme-linked immunosorbent assay (ciELISA). The Monoclonal antibody produced by the hybridoma cell was tested that there was no cross reaction with common antibiotic and structurally related compounds.In this research, MPA was transformed into artificial antigen and was immunized mice, anti-MPA monoclonal antibody was successfully obtained, which offered a good technical platform for developing MPA residual immunoassay reagent kit , which had the independent knowledge authority belonged to our country.