| Staphylococcus aureus is a common pathogen that causes suppurative disease, and can produce staphylococcus enterotoxins, which are a leading reasons of food poisoning worldwide. Among all of the food poisoning caused by enterotoxins, staphylococcus enterotoxin type A (SEA) is the most important. Therefore, establishing a sensitive, rapid detection assay to SEA is necessary for food-safety, also contributing to early diagnosis of suppurative disease caused by Staphylococcus aureus, which has important clinical and health significance. Monoclonal antibody against SEA used during the detection can effectively improve the sensitivity and specificity, but it is still not provided an effective anti-SEA monoclonal antibody in the domestic market.To adapt to the clinical detection and basic research, we designed as following study: First, to improve the sensitivity and specificity of ELISA detection, we producing anti-SEA monoclonal antibody by fusing the spleen cells taking from BALB/c mice vaccinated with SEA four times with SP2/0 myeloma cells under 50%PEG. After screening with indirect ELISA assay and limited dilution cloning, three hybridoma cell strains were obtained, and named 3C1, 3G10 and 2A10 respectively, the results of identifications of which are followed. The three hybridoma cell strains with average number of chromosomes between 87～98, can steadily secrete antibodies after culturing consecutively four months and revitalization after six months cryopreservation. The antibodies are all IgG1 isotype with k light chain, and the cross-reactivity with staphylococcus enterotoxin B (SEB) or staphylococcus enterotoxin C1 (SEC1) have not shown using ELISA assay and Western blot analysis but bind to SEA specifically. The ELISA titers of monoclonal antibodies 3C1, 3G10, and 2A10 hybridoma cell strains which get from seroperitoneum are 1:819 200, 1:51 200, and 1:12 800 separately. The ability of relative affinity of the three hybridoma cell strains which recognize the same or similar speciesspecific epitope is 3C1>3G10>2A10.An indirect competitive ELISA method based on the 3C1 monoclonal antibody detecting staphylococcus enterotoxin A in food was founded. The test conditions were optimized as followed: SEA (0.8μg/mL) were applied to 96 microtiter well plates(100/μL/well) and incubated at 37℃for 2h; the wells were blocked with 0.5% BSA in PBS at 37℃for 1 h. The titer of monoclonal antibody was 1:9 600, incubated at 37℃for 1 h with the ratio of the McAb:sample(70:30) while the reaction time of enzyme labeled antibody (1:6 000) and TMB was 30min and 10 min at 37℃. Under the qualification above, we got a standard curve, whose regression equation was y=-0.2902x＋0.8338 with the correlation coefficient r=0.9952. The detection limit of this method was 0.7311 ng/mL which can satisfy the requirement for detection SEA in foods, while coefficient variation (CV) of intra-assay and inter-assay were1.22%-13.47% and 9.31%～13.47% respectively suggested good reproducibility and accuracy. The indirect competive ELISA method based on 3C1 monoclonal antibody showed high specifity to SEA, but no cross-reaction with SEB or SEC1.We can detect SEA added in meat products at 0.8557ng/mL with the average recovery rate between the 60～120% and the CV of intra-and inter-assay〈20%.According to the results above, indirect competitive ELISA method for detection SEA can be used for clinical test, this study is a groundwork for development of kit for detecting SEA.
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