Preparation Of Murine Monoclonal Antibody Against Cronobacter Sakazakii And Analysis Of Antibody Characteristics

Cronobacter sakazakii(Enterobacter sakazakii)belongs to the family Enterobacteriaceae and has the basic characteristics and morphology of Enterobacteriaceae.The bacterium is an important food-borne pathogen that exists widely in the environment and can cause infection in infants,young children and people with weakened immunity.The morbidity rate is not high,but the mortality rate is high.Conventional detection methods for this bacteria are time-consuming,costly,and lack conventional immunological detection methods.For this purpose,monoclonal antibodies were prepared using Cronobacter sakazakii as the immunogen in this study.In this study,the formaldehyde-inactivated Cronobacter sakazakii was used as the immunogen to prepare mouse monoclonal antibodies,and the characteristics of the antibodies were analyzed.After 5 times of immunization,the spleen cells of mice whose serum titer reached more than 16 000 were collected and fused with murine myeloma cells.The positive tumor cell lines were screened by indirect ELISA.After 4subcloning,the cell lines that stably produced antibodies were named 9D10 and 2H2.The two cell lines were injected intraperitoneally into BALB/C mice to prepare two monoclonal antibody ascites.The ascites titers of the two monoclonal antibodies were measured by indirect ELISA,and the titers of the 9D10 and 2H2 antibodies were 2×10~6and 6×10~6,respectively.And was associated with Listeria monocytogenes,Salmonella typhimurium,Vibrio parahaemolyticus SH112,Bacillus cereus,Bacillus subtilis,Escherichia coli O157:H7,Klebsiella pneumoniae,Proteus vulgaris,Cronobacter turicensis,Cronobacter malonaticus,Cronobacter muytjensii,Cronobacter universalis,and Cronobacter dublinensis were not crossed.Isotype identification showed that the light chain of 9D10 antibody was kappa and the heavy chain was Ig G2a;the light chain of 2H2 antibody was kappa and the heavy chain was Ig G2a.Antibodies were labeled with horseradish peroxidase,and the competition ELISA method was used to observe the competition of the two antibody epitopes.The results showed that the 9D10 antibody was different from the 2H2 antibody epitope.In conclusion,two monoclonal antibodies against Cronobacter sakazakii with strong specificity and high titer were successfully prepared in this experiment,and the analysis of the characteristics of the antibodies can provide a basis for the establishment of a rapid detection method.