Establishment Of System For Staphylococcal Enterotoxin Ⅰ

Staphylococcus aureus is the cause of a widespread spectrum of infections in humans and different animal species.The ability to cause such a wide range of diseases may be associated with its production of a large number of extracellular toxins and other virulence factors,which includes hemolysins,nucleases,proteases,lipases, hyaluronidase,collagenase and Staphylococcal enterotoxins.These toxins cause toxic shock-like syndromes and have been implicated in food poisoning and several allergic and autoimmune diseases.Because SE is one of the most important bacteric toxin,many methods have been developed for the detection of staphylococcal enterotoxins in foods.With the researchers' work,there appear several methods such as animal detection method, immunodiffusion method,indirect hemagglutinin and biological sensor.As the most effective T cell irritant,the anti-tumor effect of SEs have been applied to clinic. Therefore,establishing Staphylococcus enterotoxins in food samples is dependent upon rapid,reliable,and sensitive immunological assay system is eagerly required. This research is aim to establish a system useful to be a quality control for the content of the SEs in the curative.we hyodermic the BALB/c mice with emulsion of purified SEI in Freund adjuvant.The fusion was performed the day after the last immunization. We fused the immune spleen cells with myeloma cells and got the hybridoma that can secrete the specific antibodies steadily.Meanwhile,we also got the polyclonal antibodies by hyodermicing the rabbits with emulsion of SEI proteinl.We can get both monoclonal antibodies and polyclonal antibodies without cross-reaction.Then we established a sensitive and rapid immunoassay system using these two antibodies for SEI primarily.Meanwhile,we also examined the accuracy rating and sensitivity. Finally,established a high performance and stable sandwich ELISA.A fusion protein GST-SEI was successfully expressed and purified from E.coli. Spleen cells obtained from mice immunized with the SEI protein were fused with the myeloma cells(sp2/0).Hybridomas were screened by enzyme-linked immunosorbent assay(ELISA) test and the stable monoclonal hybridoma were isolated by limiting dilution at least three times.At the same time,we also prepared to immunize the New Zealand white rabbits using recombinant SEI.These two antibodies were purified by two-step method using caprylic acid and amine sulphate.In this study,we established a sandwich ELISA method on testng the SEI.SEI-sepicific monoclonal antibodies were coated onto a solid-phase support to capture the SEI in the sample.And the polyclonal antibody was used to bind with another site of the SEI.SEI levels between 0.6-1.2ng/ml were consistently detectable by the ELISA,the lowest level was 0.03ng/ml.The average variation coefficient was 2.21%.