Establishment Of ELISA Method For Detecting Estradiol And Progesterone In The Urine Of Qinling Giant Pandas

Population conservation for the giant pandas is extremely urgent,plentiful breeding work has been done to solve this problem.In order to improve the reproductive rate,it’s a key aspect to confirm the female pandas’estrus cycle and pregnancy time point.At present,the estrus identification,insemination time and pregnancy diagnosis are mainly depend on detection of estrogen and progesterone levels in pandas’urine.However,there is no kit for gonadal hormone detection in giant pandas.The study aims to establish an estradiol and progesterone ELISA urine detection method,acting as a monitor to detect the change of the reproductive hormones,guiding breeding work,and improving the giant panda breeding rate.Balb/C mice were immunized by E₂-BSA and P₄-BSA.Carbodiimide method was used for synthesize coating antigen of E₂-OVA and P₄-OVA,and we also prepared and detected the estradiol and progesterone antibody.At the same time,we used the modified sodium periodate method,employing estradiol-semi-succinate and horseradish peroxidase as raw material,marking HRP to estradiol molecule;The HRP was marked on progesterone molecule by carbonization dimethylamine method,and progesterone-semi-succinate was used as raw material.Indirect competitive ELISA detection method for estradiol was established.The results were as follows:1.We synthesized coating antigen of E₂-OVA and P₄-OVA by carbodiimide method,and the composite was successfully conjugated testing by ultraviolet spectral scanning,the coupling ratio were 53:1 and 18:1.On this basis,indirect ELISA method was established for the determination of estradiol and progesterone antibody titer,and we also established the coating antigen of E₂-OVA dilution at 1:2000,the optimum dilution for estradiol antiserum was 1:2000;coating antigen of P₄-OVA dilution at 1:4000,the optimum dilution for progesterone antiserum was 1:16000;2.To acquire the estradiol and progesterone antibody,Balb/C mice were immunized by E₂-BSA and P₄-BSA.Estradiol and progesterone antibody titer were tested by indirect ELISA,1:32000 and 1:512000 respectively,which can be used for the establishment of competition ELISA method further;3.We accomplished the HRP labeled estradiol and progesterone small molecules mark,i.e.,estradiol and progesterone enzyme labeled antigen were synthesized,identifying by UV spectroscopy,the peaks of enzyme labeled antigen were different from small molecules,which proved the synthesis of the enzyme labeled antigen is successful.Meanwhile the enzyme labeled antigen was detected by direct ELISA,employing a good immunological competence;To establish an indirect competitive ELISA method for estradiol,the results showed that the detection range was 39 ng/m L-1250 ng/m L.In conclusion,the estradiol and progesterone ELISA kit established in this study provides a theoretical support and technical guarantee for detection of estradiol and progesterone in the urine of giant pandas.