Establishment And Application Of Indirect ELISA Method Based On Immunoprecipitation Antigen Of Salmonella Pullorum

Pullorum disease(PD)is an acute systemic disease of chickens caused by Salmonella pullorum(SP).The disease is mainly transmitted vertically through breeders and causes acute death of chicks within 2-3 weeks of age,causing great harm to the farming industry.The best prevention and control measure for this disease is decontamination,and the common detection method for decontamination is Serum Plate Agglutination(SPA),which is convenient and rapid,but with limited sensitivity.Therefore,this paper establishes a sensitive ELISA method by screening and identifying the antigenic proteins of the dominant strains by immunoprecipitation,which provides a good detection tool for clinical PD decontamination.First,A total of 87 SP strains were identified by collecting and bacterial isolation from samples from three yellow-feathered broiler breeder farms in Jiangsu,which were derived from 9 strains of breeder diseased tissue,31 strains of dead embryos,15 strains of bare eggs,23 strains of chicks and 9 strains of environmental samples,thus establishing an SP strain bank.Then,the bacteriophage protein complexes were obtained from the dominant strain SP001 in the strain library by mild lysis,and then the bacteriophage complexes were immunoprecipitated using immunoglobulin purified from unstaged serum after immunization.The products were analyzed by mass spectrometry and compared with the negative group to obtain a total of 112 specific proteins against SP antibody,and then 9candidate proteins were identified based on mass spectrometry scores and reproducibility.The recombinant plasmid was constructed and expressed by cloning,ligation of p ET-30a(+)expression vector,and then purified by His nickel column,and the Flavoprotein(FP)was successfully screened and attempted to be used for the establishment of ELISA method.Finally,the indirect ELISA method established based on FP recombinant protein was determined by optimizing the conditions of antigen encapsulation concentration,selection of confining solution,dilution of antibody to be detected,reaction time of antibody to be detected,concentration of secondary antibody and color development time.The highest dilution of the method for positive samples was 1:800,and the highest for similar ELISA kits was 1:200,with high sensitivity;there was no cross-reactivity with antisera of enteritis of the same group D and many common bacteria such as Escherichia coli and Bacillus cereus;the CV coefficients of the reproducibility test results were less than 10%,with good inter-and intra-batch reproducibility.In summary,this paper isolated and identified 87 strains of Salmonella pullorum,established a resource bank of yellow-feather meat breeder strains from specific regions of Jiangsu,and screened to FP antigen by immunoprecipitation method,which was used to establish an indirect ELISA method for detecting antibodies to chicken dysentery.The results showed that the method was specific,sensitive and stable between batches,and was used for monitoring 552 clinical samples,and the results showed a positive rate between 1% and 5%.The method was established to provide effective technical support for the purification of pullorum disease.