Deverlopment Of Monoclonal Antibody Against Extracellular Fatty Acid Binding Protein In Chicken And Establishment Of Double-antibody Sandwich Method

The impact of tibial dyschondroplasia(Tibial dyschondroplasia,TD)on poultry is a problem for poultry farming worldwide.There is no mature technology to detect the disease.In the early research of TD,the experimental group found that the extracellular fatty acid protein(Ex-FABP)gene was differentially expressed in the early stage of TD by gene chip technology.Therefore,the preparation of Ex-FABP monoclonal antibody was further studied.The recombinant protein Ex-FABP was purified as the immune pathogen,immunized 6 weeks old SPF BALB/C mice every two weeks。After three immunizations,the indirect ELISA was used to measure the titer,and the highest titer was selected for booster immunization.Prepare SP2/0 myeloma cell fusion.Using recombinant protein Ex-FABP and GSTA3 packets,the hybridoma cells were screened by indirect Elisa method,the positive holes with higher values were selected for sub-cloning,and 5 strains of hybridoma cell lines were obtained,named 5B7,4E4,5D12,1h6 and 4b2,and the ascites potency was up to 1 : 1011,and the subcategory identification is Ig G1.Five monoclonal antibodies were coated on the plate,and the horseradish peroxidase-labeled monoclonal antibody was an enzyme-labeled antibody.After testing,it was determined that 4E4 as the package was antibody,and the 5D12 labeled by horseradish peroxidase as secondary antibodies.Mc Ab was diluted for 1:2000 and placed overnight at 4 ℃;With whiteboard diluent containing 10% horse Serum,37 ℃ action 2h;Take 50μL of serum sample and put it at 37 ℃ for 30 min;and the enzyme-labled antibody was diluted for 1:2000,placed at 37 ℃ for 30 min;Peroxidase Substrate reaction time is 15 min.1688 clinical samples were tested by our ELISA method.The results showed that the double-antibody sandwich ELISA method had excellent specificity.The laboratory TD animal model was injected with the recombinant protein Ex-FABP animal test serum sample for Western Blot,and the SPF serum sample was set as a negative control.According to the comparison between the imaging results and the established double-antibody sandwich ELISA results,the results of Western Blot showed that the band content of Ex-FABP in serum was consistent with the value of the double-antibody sandwich ELISA method.These results indicate that the double-antibody sandwich ELISA method can be used to detect EX-FABP in serum in the laboratory.The previous study of the research team found that the early Ex-FABP gene in TD has significant up-regulated expression.In order to better study the role of Ex-FABP in TD,a monoclonal antibody against recombinant protein Ex-FABP was prepared and a double-antibody sandwich ELISA was established.The method is used to detect Ex-FABP quickly,specifically and sensitively,which lays a foundation for a better study of Ex-FABP in the later stage.