Application Of ELISA For Early Pregnancy Diagnosis Of Dairy Cows And Preparation Of Monoclonal Antibody Against PAG-1

Early pregnancy diagnosis of dairy cows is an important part of the cattle reproductive management plan,and early identification of unborn cows can create economic benefits for dairy farms.Pregnancy-Associated Glycoprotein is a diagnostic marker for early pregnancy that is closely related to maternal pregnancy.The diagnostic method based on PAG has high clinical application prospect.Obiective:The IDEXX Rapid Visual Pregnancy Test Kit compares with the rectal examination method to evaluate the practical value of the diagnostic method based on PAG in production.The eukaryotic expression system is used to obtain the fusion protein PAG-1,and the anti-PAG-1 monoclonal antibody is prepared.Verify the reactionogenicity of the monoclonal antibody.Preparation monoclonal antibody provide biomaterials for the early pregnancy diagnosis of PAG-1.Methods:1.22 cow were selected for artificial insemination.After artificial insemination for 16d,22d and 28d,PAG-ELISA kit was used to detect the pregnancy condition of the experimental cattle.The artificial insemination was performed by rectal examination for 75 days,evaluation of the application value of PAG-ELISA kit in various stages of early pregnancy based on the results of rectal examination;2.The gene synthesis method was used to obtain bovine PAG-1 gene,which was linked with eukaryotic expression vector pcDNA3.1/myc-His(-)A,and the ligation product was transferred into Escherichia coli.DH5α,digestion and sequencing were confirmed to confirm the correctness of the recombinant plasmid.The PAG-1 plasmid was transfected into HEK293 cells,and the transfected cell culture medium was purified to obtain PAG-1 protein.The SDS-PAGE and Western blot were used to identify the protein.The kit was used to detect the reactionogenicity;3.BALB/c mice were immunized with PAG-1 fusion protein,and the serum antibody titer was detected by ELISA.After reaching the fusion level,the mouse spleen was taken to obtain immune spleen cells,and hybridomas were used.Cellular methods yield cell lines that secrete anti-PAG-1 antibodies.The ascites antibody was prepared using positive cells,the antibody titer was determined,the reactivity of the monoclonal antibody with PAG-1was identified by Western Blot method,and the antibody subtype of the monoclonal antibody and the antigen binding epitope of the antibody were identified.Results:1.After artificial insemination for 75 days,5 cows were detected in the rectum,and 17 cows were not pregnant.After 28 days of artificial insemination,the PAG-ELISA kit had the highest application value.The Kappa value of the two methods was 0.681.The sensitivity,specificity,positive predictive value,negative predictive value and accuracy of ELISA kit for pregnancy diagnosis were 100%,82.4%,62.5%,100%,and 86.4%,respectively;2.The restriction endonuclease detection and sequencing alignment proved that the PAG-1 eukaryotic expression vector was constructed correctly.SDS-PAGE detected the expression of PAG-1 in cell culture medium after transfection of recombinant plasmid successfully.The expression of PAG-1 in the liquid,SDS-PAGE and Western Blot experiments were identified to a band of 41.97kDa,which was consistent with expectations.The IDEXX rapid visual pregnancy test kit proved that the PAG-1 fusion protein has a good reactionogenicity.3.After immunization,the serum antibody titer of BALB/c mice reached 2×10~5 or above,and reached the level of fusion;the hybridoma cells1F5E9C8-2 and 2F10H1D4-2 were successfully obtained,and the titer of ascites antibody was greater than 7.29×10~5;Western Blot showed 2 monoclonal antibodies react specifically to PAG-1 fusion proteins.The heavy chain subtypes of both monoclonal antibodies were IgG2a,and the light chain subtypes were all Kappa.The antigenic epitopes bound by 2 were very similar.Conclusion:1.The results of PAG-ELISA kit and rectal examination for the diagnosis of early pregnancy in dairy cows after 28 days of artificial insemination have high consistency,and PAG-ELISA method has high application value.2.Construction the PAG-1 eukaryotic expression vector successfully,purification The fusion protein PAG-1 was obtained with good purity and immunogenicity.3.Two monoclonal antibodies against PAG-1 were obtained,both of which specifically bind to the fusion protein PAG-1,and the reaction sensitivity and specificity is good,provided candidate materials for the development of diagnostic reagents for early pregnancy in dairy cows.