Development And Preliminary Of Dairy Cattle Milk Progesterone ELISA Kit

Progesterone (Progesterone, P4) is a gonadal hormone, which is the precise indicator of ovarian activity.To monitor the body progesterone content is an effective method in the prevention and treatment of reproductive disorders. The detection of progesterone content is dependent on the efficient, sensitive and specific analysing methods. In this study, a progesterone complete antigen was prepared by carbodiimide, a progesterone enzyme marker antigen was made by horseradish peroxidase, and a horseradish peroxidase marked progesterone monoclonal antibody was prepared in a simple sodium periodate method, then a competition ELISA method of cow milk progesterone (Enzyme labeled antigen method/Enzyme labeled antibody method) was established successfully. A cow milk progesterone ELISA detection kit was assembled by Enzyme labeled antigen method, and was applicated preliminarily in dairy farm.Selection of progesterone derivatives:11α-hydroxyprogesterone hemisuccinate(11α-OH-P4-HS)as a hapten was conjugated with ovalbumin(OVA) and horseradish peroxidase(HRP) by dicyclohexylcarbodiimide(DCC), respectively, to obtain complete antigen (P4-OVA) and ELISA progesterone antigen(P4-HRP). A progesterone monoclonal antibody was made by a simple sodium periodate method in laboratory, was labeled by HRP, and then a HRP-labeled progesterone monoclonal antibody could be prepared.It proved that progesterone complete antigen, enzyme labeled antigen and enzyme labeled antibody were coupled successfully by immunological method, and it was applied in the establishment of a relevant ELISA method.A direct competition ELISA method (Enzyme labeled antigen method / Enzyme labeled antibody method) was established by a self-made conjugates and progesterone monoclonal antibody to detect cow milk progesterone, and its operative conditions were optimized. The ranges of two detecting methods of milk progesterone ELISA were 0.12~ 40ng/mL for enzyme labeled antigen method, and 0.21~40ng/mL for enzyme labeled antibody method. Enzyme labeled antigen method was used to assemble the cow milk progesterone detection kit.A stablility test of all kit reagents in ELISA detection kit was admistrated by a accelerated estructive temperature for 37℃. Results showed that the kit could store at 4℃above 6 months; other test indicated that there was no cross-reaction between agent box and estradiol, estriol, and testosterone; the sensitivity was 0.14 ng/mL, intra-assay variation was 4.11%, inter-assay variation was 6.26%, the recovery rate was 93.80% ~ 107.60%, coefficient of variation was 5.65%.Cow milk progesterone detection ELISA kit was preliminary applicated in dairy farm, the accuracy of the kit was 93.3% for pregnancy and 95.8% for non-pregnancy, respectly.