Establishment Of Antibody Preparation And Detection Method Of Enterobacter Sakazakii

Enterobacter sakazakii,as a common food-borne pathogen,is ubiquitous in the environment and dairy products of infants and young children.It is easy to parasitize in the intestinal tract of humans and animals,resulting in infants and children with low immunity and animals.Disease,has a high mortality rate.In recent years,the research on the prevention and control of Enterobacter sakazakii has become a hot spot,but the lack of specific detection methods restricts the effective prevention and control of the disease.Therefore,the establishment of rapid detection method for Enterobacter sakazakii with high specificity and high sensitivity is the key to prevention and control of the disease.In this study,4 different sources of E.sakazakii standard strains were used as antigens to immunize BALB/c mice and New Zealand rabbits respectively,to prepare monoclonal and polyclonal antibodies against E.sakazakii,and to establish antibody-linked enzyme-linked Immune principle double-antibody sandwich ELISA and indirect immunofluorescence detection method.1.Monoclonal antibody against Enterobacter sakazakiiAfter 4 times of immunization with Enterobacter sakazakii,the spleen cells of mice with a serum titer of more than 16 000 were collected and fused with myeloma cells,and the selected positive hybridoma cells were subcloned.A total of 4 strains that could stably secrete resistance were selected.The monoclonal hybridoma cells of Enterobacter sakazakii were named 3A6,5B9,2B6 and EF12,respectively.The selected cell lines were injected into the abdominal cavity of mice to prepare monoclonal antibody ascites.The serum titers of the four monoclonal antibodies were above 105 measured by indirect ELISA detection method.Cross-reaction showed that the four monoclonal antibodies had good specificity and could be recognized.The six strains of Enterobacter sakazakii are different from the common enterohemorrhagic Escherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa,Vibrio parahaemolyticus,Listeria monocytogenes,and Salmonella typhimurium.No cross reaction.The results of monoclonal antibody subtype identification showed that the light chains of the four antibodies were of Kappa type,and the heavy chain except EF12 was Ig G2a and the other three strains were Ig G1.The four strains of Enterobacter sakazakii were prepared into horseradish peroxidase(HRP)enzyme-labeled antibodies by sodium iodide method,and the cross-reactivity of the four strains of antibodies was demonstrated by direct competitive ELISA detection method.There was no cross-reaction between Enterobacter monoclonal antibodies.The same 4 strains of Enterobacter sakazakii were used as mixed antigens to immunize New Zealand rabbits,and polyclonal antibodies with antibody titers above10~5were successfully obtained,which laid the foundation for the preliminary establishment of double-antibody sandwich ELISA detection method.2.Preliminary establishment of indirect immunofluorescence detection method for Enterobacter sakazakii.The flame fixation method was used to fix the bacteria to be detected on a glass slide,and an indirect immunofluorescence detection method for Enterobacter sakazakii was established.Through optimization of the test method,the best blocking solution was determined to be 1%BSA,and the blocking time was 30 min to prepare The Enterobacter sakazakii cell line 5B9 is the primary antibody,the optimal dilution factor is 1:4 000 times,and the optimal dilution factor of the FITC labeled secondary antibody is 1:20 000 times.Through the detection of simulated samples,the sensitivity of this method can reach 2.5×10~7CFU/m L,and it is compatible with common enterohemorrhagic Escherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa,Vibrio parahaemolyticus,and monocytes There was no cross-reaction between Listeria monocytogenes and Salmonella typhimurium.It shows that the established indirect immunofluorescence detection method has good specificity,and the establishment of this method provides technical support for the immunological detection of Enterobacter sakazakii.3.Establishment of double-antibody sandwich ELISA detection method for Enterobacter sakazakii.The rabbit-derived Enterobacter sakazakii polyclonal antibody was used as the capture antibody,and the mouse-derived Enterobacter sakazakii monoclonal antibody cell line 5B9 was used as the detection antibody to establish a double antibody sandwich ELISA detection method for Enterobacter sakazakii.Through optimization of the established method,it was determined that the optimal dilution ratio of the capture antibody coating was 1:4 000,the optimal blocking solution was 1%gelatin,and the optimal blocking time was 1.5 h.For detection antibodies,the optimal dilution factor is1:1000 times,and the optimal detection antibody incubation time is 1.5 h.HRP-labeled goat anti-mouse Ig G was used as the enzyme-labeled secondary antibody.The optimal dilution factor was 1:20 000 times,and the enzyme-labeled antibody incubation time was 1 hour.The minimum detection limit of the established method is 1×10~6CFU/m L,and there is no cross reaction with Staphylococcus aureus,Pseudomonas aeruginosa,Vibrio parahaemolyticus,Listeria monocytogenes,and Salmonella typhimurium.The method has good specificity.The difference between the plates and between the plates is less than 10%,indicating that the method has good stability and repeatability.This study successfully prepared Enterobacter sakazakii polyclonal antibody and monoclonal antibody,and established indirect immunofluorescence method and double-antibody sandwich ELISA detection method of Enterobacter sakazakii.The established method has strong specificity,high sensitivity and simple operation.Provide technical support for the rapid detection of Enterobacter sakazakii.