Study On The Staphylococcal Enterotoxins B Immunosensor Detection Method In Milk Products

Staphylococcal enterotoxin B (SEB) is a kind of extracellular toxins which secreted by Staphylococcus aureus, mainly in dairy and other foods with high protein content, and has a strong heat capacity, the establishment of a kind of rapid, sensitive detection method is very important. In this paper, we prepared of the SEB-specific polyclonal antibody and based immunosensor technology combined with the ideas of nano materials self-assembly to build the impedance-based electrochemical immuonsensor detection methods, the main research contents are as follows:Obtaining SEB polyclonal antibodies by immunization New Zealand white rabbits, purified antibody titer were1:80000. Indirect competitive ELISA standard curve was established, the linear range is from1ng/mL to100ng/mL; IC50is13.97ng/mL, and the SEA, SEC cross-reactivity rate is less than1.7%.By L-cysteine (L-cys) and gold nanoparticle self-assembly preparation of the immunosensor detection method, SEB antibody concentration for600μg/mL, keeping constant temperature1h, after antibody binding sites closed by BSA solution, SEB could be determined with the linearity between2ng/mL and100ng/mL; the average recovery on Dairy between87.3%and115.7%; the same concentration of SEB was repeated six times and coefficient of variation was2.35%; sensor can keep good stability at4℃for30days.Obtained polyaniline nano-gold composite film structure (PANI/Au) by the way of situ polymerization, and applied to build the immunosensors successfully. The results show that the impedance response changes of the sensor and the SEB concentration has a good linear relationship between0.1ng/mL and8ng/mL with the detection limit of0.033ng/mL (S/N=3); dairy recoveries is80.3%～112%. On this basis, using of ionic liquids to protect the PANI/Au composite membrane self-assembled immunosensor has a more favorable specificity, renewable capacity and stability.The bio-magnetic body which isolated from Taihu Lake were used in our laboratory, and conjugated of the SEB polyclonal antibody to be the magnetic body, strengthen the ability of antibody to capture SEB, was successfully prepared magnetic body immunosensor. The results show that the minimum detection limit of the sensor were further reduce to0.017ng/mL (S/D=3); average recovery of dairy were81%～118%; good stability and specificity. At the same time,it is also provides a new design ideas for the application of the magnetic body in immunosensors.The experiment established the self-assembled electrochemical immunosensor impedance detection methods,which can be applied to the detection of SEB in dairy products, providing a wealth of detailed experimental evidence and with good prospects.