Development And Application Of ELISA Kit For The Detection Of Steroid Hormones In Urine Of Female Qinling Giant Panda

Giant pandas are difficult to accurately judge the ovulation time,breed,and conceive under captive conditions,because of the species specificity.It is helpful to accurately determine the reproductive activities of fertilization,pregnancy of giant pandas,and many more,by detection of changes in the concentration of estradiol and progesterone in its urine.However,there is a lack of mature commercialization on the market to detect kits for E₂ and P₄ in the urine of giant pandas in the Qinling Mountains.The ELISA method is one of the more mature and reliable hormone detection methods,because of its simple,rapid,and sensitive.This study aims to develop an ELISA kit for the detection of E₂ and P₄ in the Qinling giant panda urine,and establish a Qinling giant panda estrus and pregnancy detection technology system to provide a theoretical basis for detecting the reproductive activities of giant pandas.The main research results are as follows:?1?The complete antigens E₂-BSA,E₂-OVA,P₄-BSA,P₄-OVA were prepared,which can be used for test animal immunization and plate coating,wherein the coupling ratio of E₂-BSA was 17:1 and the concentration was 32.35 mg/mL;the coupling ratio of E₂-OVA was 5:1 and the concentration was 2.721 mg/mL;P₄-BSA was 26:1 and the concentration was 17.889mg/mL;the coupling ratio of P₄-OVA was 9:1 and the concentration was 2.151 mg/mL.?2?The prepared complete antigen E₂-OVA or P₄-OVA was mixed with an equal amount of Freund's complete adjuvant,and fully emulsified.Then,immunoassay animals were injected into the back subcutaneously.Rabbit-derived E₂ and P₄ polyclonal antibodies were prepared.The E₂ antiserum titers were 1.28×10⁵ and 5.12×10⁵;the IC₅₀ value was3.727ng/mL;the P₄ antiserum titer was greater than 1.024×10⁶,and the IC₅₀ value was18.040ng/mL.?3?An indirect competitive ELISA method for E₂ detection was prepared,and the optimal reaction conditions were selected after optimization of the detection conditions and methods.The optimization results were as follows:coating with carbonate buffer at 4? overnight+37? for 2 h;blocking solution with 1%BSA solution at 4? overnight+37? for 2 h;competitive reaction for 1 h,reaction with secondary antibody 1 h,TMB was dissolved in 2 mg/mL DMSO.After optimization,the standard curve y=-0.1653x+1.0533?R²=0.9548?was obtained,and the kit data was improved.The results were that the cross-reaction rate of the ELISA kit with 17?-E₂-HS was 306.5%,and P₄,11?-OH-P₄-HS and T had no cross-reaction rate;the sensitivity was 112.683 pg/mL;the intra-assay coefficient of variation was 1.83%,and the inter-assay coefficient of variation was 5.63%;the accuracy was determined by the recovery rate,and the recovery rate was 90%-110%.?4?An indirect competitive ELISA method for the detection of P₄ was prepared,and the optimal reaction conditions were selected by optimizing the detection conditions and methods.The optimized results were as follows:coating with carbonate buffer at 4? overnight+37? for 2 h;blocking solution was blocked with 1%bovine serum at 4? overnight+37? for 2h;competitive reaction for 1 h,reaction with secondary antibody 1 h,TMB was dissolved in 2mg/mL DMSO.After optimization,the standard curve y=-0.3282x+1.9837?R²=0.9755?was obtained,and the performance of the kit was verified.The results were:the cross-reaction rate of the ELISA kit with 11?-OH-P₄-HS was 98.87%,and E₂,T,17?-E₂-HS had no cross-reaction rate;the sensitivity reached 309.854 pg/mL;the intra-assay coefficient of variation was 4.27%,and the inter-assay coefficient of variation was 4.98%;the accuracy was determined by the recovery rate,and the recovery rate reached 90%-110%.?5?The results of the ELISA method established by experiment were compared with those of the Academy of Forestry.The results of E₂ and P₄ were the same with the E₂concentration slightly higher than the comparison,and the P₄ concentration was basically the same as the comparison.?6?Using the ELISA method established in this experiment,urine was detected in the estrus,gestation and abortion period of giant panda II,and the changes of the giant panda hormone were analyzed.In summary,this study prepared the E₂ and P₄ ELISA kits for the Qinling giant panda urine with optimizing the optimal reaction conditions and screening out the simplest use flow,and applied it to the clinical diagnosis and pregnancy diagnosis of giant pandas.