Preparation Of Antibodies Against Bovine Pregnancy-associated Glycoprotein 19 And The Establishment Of A Triple Antibody Sandwich ELISA Method

With the diversification of demand for milk,pregnancy diagnosis is an important issue that affects the economic viability of farms and can reduce unnecessary losses.Studies have demonstrated that the levels of Pregnancy-associated glycoproteins（PAG）in maternal blood can be used to determine pregnancy.In this study,polyclonal antibodies and monoclonal antibodies against PAG19 were prepared to further investigate the use of PAG ELISA to provide new ideas for early pregnancy diagnosis.In this study,pET30a-PAG19 was expressed in prokaryotic form and immunised in chickens,and polyclonal antibodies were isolated and purified from chicken egg yolk.A recombinant pCMV3-PAG19 expression vector was constructed,the recombinant protein was expressed and isolated and purified,thereby immunising mice with the PAG19 protein as an antigen and fusing the obtained spleen cells with myeloma cells,thereby making hybridoma cells.Anti-PAG19 monoclonal antibody was produced from this positive cell line and the monoclonal antibody was identified using indirect enzyme-linked immunosorbent assay,protein blotting and other techniques.The best antibody pairings were screened using mouse monoclonal antibody as primary antibody,chicken polyclonal antibody as secondary antibody and goat anti-chicken antibody as tertiary antibody.and a preliminary triple-antibody sandwich ELISA（TAS ELISA）assay was established.The main experimental results are shown below:The pET30a-PAG19 prokaryotic protein was successfully expressed and confirmed to be produced as inclusion bodies,verifying a molecular weight size of approximately 50 k Da.pET30a-PAG19 E.coli expression conditions were explored and it was determined that the highest yield was achieved by adding 1.0 m M IPTG for 4 h at 37℃growth and OD₆₀₀=0.8,with a loading volume of 20%;bulk expression and isolation obtaining PAG19 prokaryotic protein with a purity of over 60%after purification;The gene PAG19 sequence was synthesized and the recombinant plasmid pCMV3-PAG19was constructed;the chemical transfection method and electroporation transfection method were successfully explored to transfect the target gene into HEK-293F cells,and the analysis identified that the PAG19 protein was expressed in HEK-293F cells in a soluble manner in the supernatant,and the PAG19 protein with a purity of about 90%was obtained,and the BCA measured that the concentrated PAG19 at a level of 1.1 mg/m L after concentration as measured by BCA,and the protein was tested for immunogenicity;Five strains of chicken multiple antibodies were obtained by immunisation of laying hens with the prokaryotic protein,three of which were of high potency.Five positive hybridoma cell lines were obtained by electrofusion of immunized splenocytes and myeloma cells,and after injection into the peritoneal cavity of mice,PAG19 antibody products were obtained,named:17-10H-1,17-5H-3,18-1H-5,23-9-3C-2,24-1D-1;antibody potency was up to 1×12800;antibody subtypes identified:17-10H-1,17-5H-3,18-1H-5 are IgG2a,24-1D-1,23-9-3C-2 are IgG2b;using TAS ELISA pairing,the optimal concentration of encapsulated monoclonal antibody is 2μg/m L,the concentration of antigen is 5μg/m L,the optimal concentration of multiple antibody is 0.125μg/m L;the optimal incubation time for both antigen and antibody is1 h.Preliminary exploration shows that The method has good sensitivity and specificity.