Immunomagnetic Beads Technique For Concentration Of Salmonella Enteritidis And Staphylococcus Aureus

The salmonella is one of the main pathogens of human food poisoning, pathogenic to the human being and animal, causing a variety of human and animal diseases, In the world, meat and eggs contaminated by salmonella causing food poisoning is still account for a large proportion.Staphylococcus aureus can cause multi-tissue infections, such as bacteremia, endocarditis and pneumonia. Food poisoning caused by the Staphylococcus aureus is living in the second or third, the major source of contaminated, secondly only to Salmonella.Immunomagnetic beads is coated with associated immune ligand (-NH2,-COOH, et al.) of magnetic particles, utilize antigen-antibody reaction of specificity to isolate the target material from the mixture, features has a high degree of specificity of immune response, operation simply, does not require expensive equipment. Food of Salmonella is mainly used in the test isolation and culture methods, as less pollution, conventional tests carried out by bacteria must be cultured to increase the detection rate, which takes longer, the detection rate of less difficult to carry out immediate on-site rapid testing. In this study, immunomagnetic separation and antibody preparation, contaminating bacteria in the very little samples, enhance Salmonella and Staphylococcus aureus in the detection rate and shorten the test time for the market, providing more convenient quarantine rapid test methods.1. Preparation of rabbit/chicken polyclonal antibody anti-Salmonella enteritidisImmunogen of Salmonella enteritidis, emulsified with Freund's adjuvant to immunize rabbits and chickens, obtain anti-SE serum respectively, depurate saturated ammonium sulphate, G-200 column purification and octanoic acid-ammonium sulfate purification. Obtain rabbit anti-SE and chicken anti-SE of PcAb, for the IMS-XLD method to establish offering the material foundation.2. Immunomagnetic beads technique concentration of Salmonella enteritidis and Immunomagnetic beads storageThe synthesis of the magnetic particles utilize chemical co-precipitation method, particle diameter is about 30nm-100nm, and its surface modification of silane and glutaraldehyde. The modified magnetic particles was coated by antibody of anti-SE, beads and antibody coupling conditions were confirmed, such as the temperature of coated, reaction medium, etc. Utilize 200μL immunomagnetic beads mix with culture solution of bacteria about 30 min, that can separate Salmonella enteritidis from culture solution, sensitivity is 3cfu/mL. Compared the immunomagnetic beads separation technique to bacterial culture, immunomagnetic beads separation technique is better than bacteria.Two ways of freeze-dried and liquid preserved to storage the beads, in different con-ditions and different time, test adsorption capacity to determine the way of preserva-tion. Adsorption capacity of Lyophilization magnetic beads reduce, so do not use this method. In the liquid preserved group, the addition of protective agents immunemag- ne tic beads group absorption capacity of bacteria better than unprotected control agents, so use 0.1‰thimerosal mixture with 1% sucrose solution as the preservation of the immune beads.3. Immunomagnetic beads technique concentration of Staphylococcus aureusThe synthesis of the magnetic particles utilize chemical co-precipitation method, establish immunomagnetic beads method concentration of Staphylococcus aureus. Exper-imental results showed that: utilize 100μL immunomagnetic beads mix with culture solu-tion of bacteria about 15 min, that can separate Staphylococcus aureus from culture solu-tion, sensitivity is 5cfu/mL. ELISA is combined with IMS-ELISA method attempts were not successful, further research to be in the future.