Establishment Of Sandwich ELISA Method For Detecting Enterococcus Faecalis Double Antibody In Milk

Mastitis in dairy cows poses a great threat to the safety of dairy products in Inner Mongolia,China.Studies have shown that Enterococcus faecalis is the number one pathogenic bacterium causing mastitis in dairy cows,while the source milk samples also carry Enterococcus faecalis.Enterococcus faecalis is a commensal bacterium in the intestinal tract of humans and animals,and it is an opportunistic pathogen that can cause infection when the immunity of humans or animals is low.The transfer of E.faecalis to finished milk through raw milk can cause a variety of complex human diseases,which are difficult to cure,often recurring,and persistently infected.Therefore,the establishment of efficient and specific detection methods is a pressing issue today.How to detect pathogenic bacteria in an accurate and timely manner plays an important role in ensuring the safety of dairy products.This study was carried out to prepare polyclonal antibodies against Escherichia faecalis Esp protein and to establish a double antibody sandwich ELISA method for the detection of Escherichia faecalis in bovine milk to achieve rapid and accurate detection of bovine milk quality.In this experiment,the sequence of Enterococcus faecalis esp gene published on Genbank with sequence number AY032999.1 was used as a template for cloning and sequence analysis of esp gene,and the target DNA strip with sequence length of 891bp was successfully cloned for the construction of pET-30a-esp/BL21 and the efficient expression of Esp protein.Two sets of polyclonal antibodies against Esp serum were prepared by rabbit and mouse immunoassay,and the potency of the two sets of polyclonal antibodies was determined by indirect ELISA after purification,and the potency of the rabbit-derived antibody was finally determined to be 1:1600 and that of the mouse-derived antibody was 1:3200 with good antibody specificity.Subsequently,the reaction conditions were optimized on the basis of the original double antibody sandwich ELISA method,and the optimized double antibody sandwich ELISA method was established for the detection of Enterococcus faecalis in bovine dairy products,and the final decision was made to use rabbit-derived antibody as the encapsulated antibody,which was diluted at 1:200 at 4℃for 12h,and then closed at 37℃for 60min,and the antigen was added at 37℃for 60min.Mouse-derived antibody was used as the detection antibody,diluted at 1:100 at 37℃for 60min,and HRP-goat anti-mouse IgG was selected as the enzyme-labeled antibody,with the optimal reaction conditions of37℃,60min and the optimal dilution of 1:8000,after which the reaction was terminated by adding TMB chromogenic solution and avoiding light for 10min.The standard curve of the ELISA method was developed,and 36 negative samples were tested using the method.The Cut-off value of the method was calculated to be 0.382,and good results were obtained for the accuracy,reproducibility and specificity of the method.The method was successfully used to detect emulsion contaminated with Enterococcus faecalis in actual cow’s milk samples,and then the sensitivity of the method was investigated in the enriched cow’s milk samples,and the results were obtained to detect cow’s milk with bacterial concentration of 1×10~4 CFU/m L.The double antibody sandwich ELISA assay established in this study is accurate,stable,specific and sensitive,and can achieve rapid and effective detection in the actual detection of cow’s milk.