Development Of Quantum Dots Fluorescence Test Kit For Staphylococcal Aureus Enterotoxin A

Staphylococcus aureus is an important pathogen causing suppurative diseases,and can produce staphylococcus enterotoxins,which are a leading reasons of food poisoning worldwide.Anong all of the food poisoning caused by enterotoxins,staphylococcus enterotoxin type A(SEA)is the most important reason.Therefore,establishing a sensitive,rapid detection method to SEA is necessary for food-safety caused by Staphylococcus aureus.Monoclonal antibody against SEA used during the detection can effectively improve the specificity.In order to satisfy the needs of basie research,and explore the rapid detection assay of SEA,the research is based on the platform provided by Vazyme Biotech Co.,Ltd and Vazyme Medical Co.,Ltd.Firstly,Monoclonal antibody against SEA was prepared,then a quantum dot test kit for qualtitative detection was developed.In The technique,qUantum dots were used as tracers,which raplace traditional fluorescent markers and have the advantages of fast,sensitive and quantitative detection.The research contains three parts including the preparation of monoclonal antibody against SEA,the development of quantum dot test kit and the validation of the performance.The main contents of the research are as follows:Firstly,Balb/c mice were immunized with the recombinant protein of SEA,and indirect ELISA assay was used to detect serum titer,and the titer was beyond 6400.Monoclonal antibodies were prepared by inducing ascites in vivo.Finally,two monoclonal antibodies named 2C2 and 7B5 were obtained,the antibody has specific ability to recognize SEA,and the types of the two strains are all IgG1 isotype.Indirect eompetitive ELISA assay was used to detect the titers of two monoclonal antibodies.The titer of 2C2 and 7B5 was 5.12×106 and 2.56x106 respectively.At the same time,the specificity of McAbs was evaluated by indirect competitive ELISA and Western blotting,the results showed that the specificity of the antibody was good.Secondly,the kit was developed by the immune chromatography technology combining with the Quantum dot labeled with monoclonal antibody,in order to improve the sensitivity and anti-interference ability of the kit,the process and raw materials of the kit were optimized.During the optimization process,the concentration of the secondary antibody coated with C line was determined to be 1.5 mg/mL,the concentration of 7B5 coated on the T line was 0.5 mg/mL.The antibody and QDs were chemically conjugated,and the complex was diluted with the buffer containing 0.5%BSA at the ratio of 1:50.Millipore HFC 90 was chosen as the best coating carrier for NC membrane.The coating buffer contained 1%sucrose,blocking reagent contained 1%sucrose and 1%casein.Lastly,the performance of the kit was verified and validated.The sensitivity of the kit was l.0 ng/mL,the linear range was 1.0-100 ng/mL.The standard samples of different concentrations and types of enterotoxins were detected by the kit,the results show that there was no obvious cross reaction and the specificity was good.Moreover,the repeatability of high and low values of SEA standard solution was tested,thhe results show that the coefficient of variation(CV)was less than 5%,and the precision of the kit was good.The coefficient of variation between the kits stored at 2-8? and the kits accelerated aging at 37°C for 20 days was less than15%,so the stability of the kit was good.The kit was applied to the detection of food samples,and the reclaiming experiment of adding samples was carried out on the kit.The results show that the average recovery of SEA in milk was 95.7%,the recovery rate of the kit ranged from 60%to 120%,it was fully proved that the kit has reliable test results.