Preparation Of A Monoclonal Antibody Against A Highly Conserved Epitope Of PEDV S Protein And Establishment Of Its Detection Method

Objective: To prepare a monoclonal antibody against the conserved epitope of the S protein of porcine epidemic diarrhea virus,to provide a material basis for the immunological detection method of PEDV,and to establish a double antibody sandwich ELISA detection method for detecting PEDV.Methods:(1)Preparation of immunogen: Two different B cell linear epitope prediction methods were used to predict the epitope of the S protein of PEDV,and the dominant epitopes predicted by the two methods were initially screened.At the same time,the sequence alignment of the S protein genes of 27 PEDV virus strains was carried out,and the S protein gene was used to model the S protein homology,and the antigen epitopes that were both highly conserved and in favorable structural positions were further screened.The selected epitopes were synthesized and conjugated with BSA as immunogens.(2)Preparation of screening antigen: Use Vero cells to culture the virus and purify the virus by ammonium sulfate precipitation,sucrose density gradient centrifugation and PEG6000 precipitation.The purification effect was verified by ELISA and SDS-PAGE using known monoclonal antibodies against PEDV.The antigen with good purification effect is used as the screening antigen.(3)Immunize mice with the synthesized antigens separately,select a batch of mice with the most ideal titer for continued immunization,and fusion after 4immunizations,and screen hybridoma cells by ELISA method,and use the supernatant of positive hybridoma cells to determine After subtype identification and specificity verification of the secreted antibodies,ascites monoclonal antibodies are prepared.After the antibody is purified,its concentration and titer are detected,and the antibody is labeled with HRP and the labeling conditions are explored to establish a double antibody sandwich ELISA detection method.Results: Three theoretically ideal highly conserved epitopes were screened out.After 3immunization,the titer was determined.The immune titers of No.1 and No.2 epitopes were 4000～8000,while the efficacy of No.3 epitope The price reached close to 16,000.Select peptide 3 to immunize mice for the preparation of monoclonal antibodies.Two strains of monoclonal antibodies that can stably secrete PEDV were screened.The ascites-type antibody titer was 1.3 mg/ml after purification,and the titer was approximately Is 6400.When HRP-labeled antibodies,the molar mass ratio of HRP:Ab is 4:1 and the effect is ideal.And use 2C2 antibody to establish a self-paired double-antibody sandwich ELISA detection method.Conclusion: Using the epitope prediction of the S protein,multiple sequence alignments and the immunogens screened out by homology modeling,a monoclonal antibody against the highly conserved epitope of PEDV was successfully prepared,and the antibody itself can be paired and used for the establishment of double antibody sandwich ELISA.The preparation of the antibody provides an important material basis for PEDV immunological detection methods.