Preparation Of Anti-Progesterone Monoclonal Antibodies And Development Of Enzyme Immunoassay For The Detection Of Progesterone Content

This paper reported the preparation of Progesterone-McAb and the development of enzyme immunoassay for the detection of progesterone content.Progesterone is a kind of steroid hormone.it,s content raise up with gestation in cow,s milk.so that the variation of the content of progesterone should reflect whether the milk was gestate. It is very important to establish a kind of credible, sensitive and quick assaying method.Progesterone was a hapten. To prepare antigen,11ɑ-OH-progesterone was activated, and then 11ɑ-OH-P was conjugated to carrier proteins KLH, BSA and OVA by carbodiimide. P-KLH and P-BSA were used for the immunization of mice and P-OVA was used as the coating antigen for the ELISA study. Both antiserums of P-KLH and P-BSA could bind specifically with Progesterone. The anti-progesterone antibody titer of antiserum immunized with P-BSA was higher than that with P-KLH.Fore hybridoma secreting monoclonal antibodies against Progesterone were achieved by injection of BALB/c mice with P-BSA. Three hybridoma cell lines of 1F6,2G6 and 2A11 were obtained using P-OVA by indirect ELISA, and cloned by limited dilution method for three times, whose titers of cultivate medium were 1︰1600, 1︰800, 1︰400 respectively. These three McAbs were characterized for the cross reactivities, specificities, affinity abilities vs. progesterone. The titer degree of ascites fluid of these three McAb was 1︰1×106～1︰8×104. These three McAbs showed <0.01% cross reaction with the other steroid hormone. Relative affinity showed 1F6>2G6>2A11. The subtype of 1F6 was IgG2b,2G6 and 2A11 were both IgM.Progesterone was to be labelled with HRP. HRP-P could bind with P-McAb specifically. The samples was added in the ELISA plat wells which were coated with P-McAb and blocked with 1% gelatin. Progesterone in the samples could bind with P-McAb. After washing, the HRP-P was added and incubated for 15min at 37℃. After the plat was washed again, OPD-H2O2 was added and the reaction was terminated by adding 2M H2SO4 in 15 min. The OD490nm values was read at 490nm in a microplat reader. The contraction of P-McAb and dilution of HRP-P were determined. The standard curve was obtained. This immunoassay provided a analytical method for the detection of progesterone content. It could be used in preparation of progesterone ELISA kit,and basis of exploitation of colloidal gold test paper.