Establishment And Application Of A Double-antibody Sandwich ELISA Method For Staphylococcal Enterotoxin K

Staphylococcus aureus（SA）is gram-positive cocci and an important foodborne pathogen,which can cause food poisoning by producing staphylococcal enterotoxin（SEs）.90%of staphylococcal food poisoning are caused by classical enterotoxins,and the remaining 10%are caused by new staphylococcal enterotoxins or staphylococcal enterotoxin-like（SEls）.The sek gene encoding SEK protein has a high detection rate in foodborne Staphylococcus aureus strains,indicating that SEK protein may be an important potential pathogenic factor for food poisoning.In this study,murine monoclonal antibody and rabbit polyclonal antibody were prepared by cloning and expression of sek gene,and a double-antibody sandwich ELISA method was established to detect SEK protein.The influencing factors of SEK protein production in food were analyzed from three aspects:culture,temperature and time.The specific research contents are shown as follows:1.Preparation of monoclonal antibodies against SEK proteinUsing Staphylococcus aureus strain SA1128271 stored in laboratory as template,sek gene was amplified by PCR,and prokaryotic expression plasmids sek-p ET-32a（+）and sek-GST-6p-1 were constructed.His-SEK recombinant protein and GST-SEK recombinant protein with concentration of 0.54 mg/m L and 3.48 mg/m L were obtained after induced expression.His-SEK protein was mixed with rapid immune adjuvant to immunize female BALB/c mice,and cell fusion was performed after 3 days of enhanced immunity.After indirect ELISA screening and subclonal culture,4 cell strains secreting monoclonal antibodies against SEK protein were obtained,which were 8D10,4B11,8B3,8B2.The monoclonal antibodies obtained were identified by Western Blot,and it was confirmed that all 4 monoclonal antibodies could react specifically with SEK recombinant protein.Subtype analysis showed that the light chain type of the 4 monoclonal antibodies wasκ,and the heavy chain were different IgG subtypes.According to the titer of ascites,8B2 cell strain was selected to produce ascites in large quantities,and the concentration of purified monoclonal antibody was 1.56 mg/m L.The titer of monoclonal antibody was100×2¹².2.Preparation of polyclonal antibodies against SEK proteinNew Zealand white rabbits were immunized with His-SEK protein mixed with rapid immune adjuvant.After the sixth immune to New Zealand white rabbits serum,blood collection and separation by antigens purified polyclonal antibody affinity chromatography purification method.The polyclonal antibody was identified by Western Blot,and it was confirmed that the polyclonal antibody could react specifically with the SEK recombinant protein.Polyclonal antibody concentration was 2 mg/m L.The titer test showed that the titer of polyclonal antibody was 100×2¹².3.Establishment of a double-antibody sandwich ELISA method for the detection of SEK double antibodyA double-antibody sandwich ELISA method for the detection of SEK protein was established using a monoclonal antibody as the capture antibody and a polyclonal antibody as the detection antibody.The optimal coating concentration of monoclonal antibody was 0.5μg/m L,the optimal dilution of polyclonal antibody was 1:8000.The regression equation was y=0.0048x+0.0948 with a correlation coefficient R²=0.9994,and the detection limit was 7.20 ng/m L.The method of SEK double antibody sandwich ELISA did not cross-react with SEA,SEB,SEC and SEY,and the specificity was good.4.Exploration of the factors influencing SEK protein productionThe SEK expression of Staphylococcus aureus in LB media,milk and pork cultures at 37℃and 25℃was detected by growth curves assay,real time PCR and sandwich ELISA,and its influencing factors were analyzed from three aspects:culture,temperature and time.The results showed that the stable period of Staphylococcus aureus in LB media and milk was earlier than that in pork.Expression of the sek gene in milk and pork was highly expressed twice during the growth period of Staphylococcus aureus.SEK protein expression of Staphylococcus aureus increased sequentially with time in all three cultures.Compared with 25℃,37℃was more likely to produce SEK protein.Compared with pork,Staphylococcus aureus in milk is more likely to produce SEK protein.The SEK protein produced by Staphylococcus aureus in pork was higher than 20 ng/ml when cultured at37℃for 24 h,suggesting that Staphylococcus aureus in pork at this time had a risk of food poisoning.This study provides technical support for the surveillance of Staphylococcus aureus food poisoning and provides a basis for the formulation of SEs prevention and control measures.