Study On Rapid Detection Technology Of Early Pregnancy In Equine Animals

Object: The continuous updating of diagnostic methods for early pregnancy of equine animals is the basis for the development of equine animal husbandry.The updating of diagnostic methods for early pregnancy not only increases the economic income,but also better supplements nutrition and effectively protects pregnant animals,increases production efficiency and reduces accidental abortion.The aim of this study was to establish a rapid diagnostic technique for early pregnancy in equine animals.Methods: 1.Establishing an Enzyme-Linked Immunosorbent Assay(ELISA)detection kit of Pregnant Mare Serum Gonadotropin(PMSG)by double antibody sandwich method;2.The citric acid reduction method and 4-(2-hydroxyethyl)-1-piperazineethanesulfonicaci(HEPES)one-step method were used to prepare gold nanoparticles with different colors and shapes.Based on the principle of double antibody sandwich method,the lateral flow immunochromatographic technique of PMSG was established.Results: 1.PMSG was detected by ELISA double-antibody sandwich method.The results showed that the optimal reaction conditions were as follows: The optimal concentration of monoclonal antibody was 8.5μg/m L;The optimum working concentration of enzyme antibody is 3.5 μg/m L;The best coating diluent is CB;The best sealing time is 4 hours;The best antigen reaction time is 60 minutes;The best reaction time of labeled monoclonal antibody is 60 minutes;The best time of substrate action is 10 minutes.2.The established ELISA double antibody sandwich method has good specificity;Sensitivity is 25 IU/m L;Good repeatability;50 IU/m L was determined as the critical value of pregnancy and non-pregnancy.3.The lateral flow immunochromatography technique of PMSG was established: the lateral flow immunochromatography test strip of PMSG was prepared by double antibody sandwich method,and the optimized results were as follows:(1)Using trisodium citrate as reducing agent and stabilizer to prepare gold nanoparticles,a wine-red colloid with bright color and no precipitation was obtained,and a surface plasmon resonance peak appeared at 520 nm,with a narrow peak shape and a half-width of about 30 nm,spherical gold nanoparticles with a particle size of about 20 nm.After optimization,it is determined that the preparation method of flower-shaped gold nanoparticles is a one-step method.Chloroauric acid is reduced by HEPES at room temperature,and the obtained gold nanoparticle solution is bright sea blue,uniform in color,free of stratification,floating objects and precipitation.The surface plasmon resonance peak appears at 594 nm,which is consistent with the color displayed by the solution.The peak shape is narrow,the half-peak width is about 80 nm and the average diameter is 50 ± 5 nm.(2)The optimal stability of gold nanoparticle labeled antibody is that 4.8 μL labeled antibody 9F6 is added into 1 m L gold nanoparticle solution;The best p H value of colloidal labeled antibody is 8.0;GF60-S glass fiber membrane is the best sample pad material;The sample pad treated with 0.02 M Tris-HCl buffer containing 10% BSA,1%Tween-20 and 10% sucrose had the best effect.33 Glass type glass fiber film is the best bonding pad material;Adding 5 μL sealant to the bonding pad is the best;Millipore 180 nitrocellulose membrane is the most suitable;The best condition of T-line coating anti-PMSG monoclonal antibody 1H9 is monoclonal antibody: coating diluent=8:2.4.The specificity of the established PMSG lateral flow immunochromatographic strip was good.The sensitivity of the red strips was 50 IU/m L and that of the blue strips was 25 IU/m L.Repeatability and stability are good.Conclusion: 1.In this experiment,an ELISA double antibody sandwich method was established,and its reaction conditions were optimized,so as to obtain a specific,reproducible and sensitive ELISA method for detecting PMSG.2.The solution of wine red spherical gold nanoparticles prepared by trisodium citrate reduction method has uniform particle size,good stability and strong monodispersity;The blue flower-shaped gold nanoparticle solution prepared by HEPES one-step method has uniform particle size,good stability and good dispersibility.3.In this experiment,two kinds of PMSG lateral flow immunochromatography test strips with different colors were prepared.The red and blue test strips have good specificity,but the sensitivity of the blue test strip is higher than that of the red one.In this experiment,the detection method of PMSG was established by lateral flow immunochromatography,which laid a foundation for simple,rapid,convenient,high-sensitivity and low-cost early pregnancy detection of equine animals.