Preparation Of Two Monoclonal Antibodies Against Food-borne Pathogens From Cow Milk And Establishment Of Immunomagnetic Beads For Isolation

Listeria monocytogenes and Salmonella typhimurium are common food borne pathogens,which can contaminate a variety of foods and cause serious losses to food safety.Both pathogens are widely distributed in the environment,and the mortality rate of adults and the elderly and infants with low immunity after infection is high.Listeria monocytogenes can cause miscarriage in pregnant women and cause fetal or neonatal pneumonia and meningitis.Salmonella typhimurium can cause systemic illness such as vomiting,gastroenteritis and severe diarrhea,which can lead to intestinal obstruction,gastrointestinal bleeding and even death if left untreated.Molecular detection and microbiological detection are the most common methods for these two kinds of bacteria,but their cost is high and time consuming is long.Therefore,this study established a rapid immunomagnetic separation and enrichment method for Salmonella typhimurium and Listeria monocytogenes,which reduced the enrichment difficulty of the two food-borne pathogens and provided a new technical scheme for the detection of the two food borne pathogens.In this study,three whole strains of Listeria monocytogenes were inactivated by formaldehyde as immunogens to prepare monoclonal antibodies against Listeria monocytogenes,and the characteristics of antibodies were analyzed.A total of 8 monoclonal antibodies were screened.The titers of the antibodies were above1×10~6 and the specificity was good.The light chains of the 8 antibodies were all Kappa type,and the heavy chains were all Ig M type.Three Salmonella typhimurium strains were inactivated with formaldehyde as immunogen to prepare monoclonal antibodies against Salmonella typhimurium.A total of 4 monoclonal antibodies were screened out.The titer of the antibodies was above 4×10~6 and the specificity was good.The light chain of the four antibodies was Kappa type,and the heavy chain was Ig G1 type.The two monoclonal antibodies prepared in this study had excellent properties,which laid the foundation for the later establishment of the immunomagnetic bead separation method of Listeria monocytogenes and Salmonella typhimurium.Listeria monocytogenes and Salmonella typhimurium monoclonal antibodies were conjugated with carboxyl magnetic beads to prepare immunomagnetic beads for enrichment of target bacteria.In this study,the magnetic bead coupling buffer,magnetic bead diameter and different capture times were optimized.The results showed that the optimal conditions for Listeria monocytogenes immunomagnetic beads were as follows:coupling buffer was MEST(0.025 M,p H 7.0),the optimal diameter of magnetic beads was 750 nm,and the capture rate was the highest at 45 min.The optimal conditions for Salmonella typhimurium immunomagnetic beads were as follows:coupling buffer was MEST(0.025 M,p H 7.0),the optimal diameter of magnetic beads was 180 nm,and the capture rate was the highest at 45 min.Specificity analysis showed that the capture rates of the two kinds of immunomagnetic beads to target bacteria were above 90%,while the capture rates of other common food borne pathogens were below10%,indicating that the two kinds of immunomagnetic beads prepared in this study had excellent capture rates and good specificity.It lays a foundation for the later detection of Listeria monocytogenes and Salmonella typhimurium in milk.