| Staphylococcus aureus is a major human and zoonotic pathogen widely spreading in the nature. It can produce staphylococcal enterotoxins (SEs) and induce food poisoning. Detection of SEs is listed in food safety inspection regulations with the improving of people's awareness about food safety. It is necessary to establish a rapid and sensitive method for detection of SEs to insure food safety.Methods for detection of SEs have been mainly based on zoology test, immunological methods, polymerase chain reaction, biosensors, and technique of superantigens. Among these methods, immunological methods are convenient, rapid, sensitive and specific. It is a widely-used technology for detection of SEs. Monoclonal antibody (McAb) has the benefit of high purity and specificity, and can be put into full scale production. If it is used for immunological detection of SEs, it can improve the level of specificity and sensitivity in detection. Monoclonal anibodies used in commercial ELISA kits for detecting SEs are mainly pepared by hybridomas technique and immunized with single antigen, and it can only detect its related toxin. At present, serotypes of enterotoxin are above 20 types, and staphylococcal food poisoning is not usually only caused by a single toxin. In actual testing process, sometimes there is no need to identify serotypes of enterotoxin, it is important to determine whether enterotoxins exist. So it will greatly reduce detecting workload when a variety of toxins can be simultaneously determined in one reaction. Therefore, if monoclonal antibody against common antigen determinants shared by multiple SEs can be prepared by the method of simultaneous immunization, it is possible to detect a variety of enterotoxins in one reaction.In this paper, through hybridomas technique, one hybridomas cell, designated 3F2, was obtained when Staphylococcal enterotoxin A (SEA) and B (SEB) were used as immunogen by the method of simultaneous immunization. Hybridomas cell 3F2 can produce antibody against both SEA and SEB. At the same time, the other hybridoma cell was obtained following the same procedure with mouse immunized by SEA, which was designated 3C12. Two monoclonal antibodies were obtained by injection 3F2 and 3C12 into BALB/c mice, respectively. Characteristics of two antibodies were determined by ELISA method. Potential application for 3F2 to detection SEs in dairy products was evaluated by indirect competitive ELISA. Results were as followed.1 Preparation and analysis of polyclonal antibody against SEA-SEBBALB/c mice (No.1～3) were simultaneously immunized with equal amount of SEA and SEB, immunizing dose of each mouse was 20μg every time (SEA and SEB were 10μg, respectively). After a week' immunization, mice were bled and antisera were obtained. Characteristics of antisera were analyzed by an indirect ELISA. There were obvious antibodies produced at 21st day, and mice had the highest titer of antibodies at 49th day. After the fourth immunization, the titer of antisera from mouse No.2 was 4.5×104 for SEA, and 5.0×104 for SEB; sensitivity of antisera was 81.3 ng/mL for SEA and 62.4 ng/mL for SEB. Antisera had high specificity against both SEA and SEB. It had no cross reaction with staphylococcal enterotoxin Cl (SEC1), bovine serum albumin (BSA) and ovalbumin (OVA).BALB/c mice (No.4～6) immunized with SEA followed the same procedure, and immunizing dose of each mouse was 10μg every time. Characteristics of antisera were analyzed by an indirect ELISA. There were obvious antibodies produced at 21st day, and mice had the highest titer of antibodies at 49th day. After the fourth immunization, the titer of antisera from mouse No.4 was 5.0×104 for SEA; sensitivity of antisera was 73.9 ng/mL for SEA. Antisera had high specificity against SEA. It had no cross reaction with SEB, SEC1, BSA and OVA.2 Preparation and analysis of monoclonal antibody against SEA-SEBMcAb was produced by fusing the spleen cells taken from mouse No.2 with SP2/0 myeloma cells after the mouse was immunized by multi-antigen (SEA and SEB). By screened using the method of an indirect ELISA and subcloned by limiting dilution assay, one hybridoma cell was obtaind, designated 3F2. It secreted stable antibodies and reacted with both SEA and SEB. The other hybridoma cell, designated 3C12, was obtained following the same procedure with mouse No.4 immunized by SEA. Two monoclonal antibodies were obtained by injection of 3F2 and 3C12 hybridoma cells into BALB/c mice, respectively. To ananlyze and contrast characteristics between two antibodies, the McAb's titer, sensitivities, specificities, affinities, the chromosomal number of hybridoma and stability of antibody were determined by ELISA method, respectively. Results indicated that the titer of ascites from hybridoma 3F2 against SEA was 4×105, sensitivity was 133.2 ng/mL, affinity was 8.18×107 L/mol; the titer of 3F2 against SEB was 1×105, sensitivity was 82.5 ng/mL, affinity was 5.32×107 L/mol. It had no cross reaction with SEC1, BSA and OVA. The titer of ascites from hybridoma 3C12 against SEA was 2×105, sensitivity was 115.9 ng/mL, and affinity was 6.76×107 L/mol. It had no cross reaction with SEC1, BSA and OVA and its cross reaction with SEB was weak.Both hybridoma 3F2 and 3C12 had chromosomal number of about 100 after chromosome analysis, and it is similar with the number of mixture of spleen cells and SP2/0 myeloma cells. This indicated that two hybridoma cells obtained were fusion of parent cells. Two hybridoma cells had stable antibody titer when they had passed down serial generations, frozen and thawn in three months, which idicated that they had a good stability.3 Initial application of McAb 3F2 against SEA-SEB for SEs detecion in dairy productsDairy products easily contaminated with SEs were selected for this study. Effect of detection SEs in food using McAb 3F2 obtained by simultaneous immunization was evaluated. (1) Dairy products uncontaminated with SEs were spiked with SEA or SEB, respectively. McAb 3F2 and 3C12 were used for detection SEs in these samples. Average recoveries of spiked samples were assayed by indirect competitive ELISA. McAb 3F2 had good recoveries for both SEA and SEB, average recoveries of spiked milk were from 88.3% to 103.5% for SEA and from 86.2% to 101.6% for SEB. Average recoveries of spiked milk were from 86.7% to 98.1% for SEA with McAb 3C12. (2) SEA, SEB and SEC1 were mixed with various amounts, spiked into milk samples, and then SEA and SEB were determined using McAb 3F2 by icELISA. It showed that both SEA and SEB could be determined without interfered by SEC1. Average recoveries of spiked milk were from 70% to 120% for both SEA and SEB. (3) Milk and yogurt samples uncontaminated by SEs were inoculated with SEA or SEB secreting strains in manner of single and simultaneous inoculation. After incubated 36～48 hours at 37℃, SEs were detected using McAb 3F2 by icELISA, both SEA and SEB could be detected.
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