Establishment Of AlphaLISA Detection System For 11 Toxins

Biotoxins could be roughly divided into fungal toxins,bacterial toxins,plant toxins,animal toxins and Marine biological toxins.In this study,11 biotoxins were selected,including 5 major enterotoxins of staphylococcus aureus(SEA,SEB,SEC,SED,SEE),botulinum toxin type A(Bo NT-A),clostridium perfringens endotoxin(CPA),clostridium perfringens toxin(ETX),acacia toxin(AT),ricin(RT)and a small molecule of the toxin T-2.All of these 11 toxins could cause human poisoning events.Once a poisoning event occurs,the most important thing was to quickly detect the type and approximate dose of toxic toxins.In order to improve the detection efficiency of these 11 toxins,Alpha LISA detection system for these 11 toxins was established in this study.The study was divided into two parts.Alpha LISA detection system for 10 toxins(SEA,SEB,SEC,SED,SEE,Bo N-A,CPA,ETX,AT,RT)were established in the first part.The second part of the study was that a competitive Alpha LISA detection system for small molecule T-2 toxins.Alpha LISA technology was a rapid detection method,which required small sample volum,was easy to operate,taked less time and had high accuracy.In the establishment of the Alpha LISA detection system for these 11 toxins,we firstly screened the most suitable antibody for each toxin by ELISA technology,and carried out Alpha LISA pre-test with the screened optimal antibody to optimize the reaction conditions and determine the optimal detection scheme for each toxin.Then,11 purified toxins were tested for Alpha LISA,the standard curve of toxins was drawn,and the detection limit,specificity and repeatability of each toxin were evaluated.Subsequently,SEA simulation samples,Bo NT-A simulation samples and T-2 toxin simulation samples were designed and prepared to evaluate the sensitivity of Alpha LISA detection system.We also collected clinical samples of botulinum toxin to evaluate the Alpha LISA test system of botulinum toxin and to compare the correlation between test results and clinical symptoms.The results showed that the sensitivity of all the 11 toxin Alpha LISA detection systems we established was less than 1ng/m L,and the sensitivity of SEB was up to 25pg/m L.With good specificity,the established detection system for Alpha LISA of 11 toxins did not cross-react with other toxins.The repeatability was good,and the variation coefficient between batches and within batches was less than 10%.In the first part of this study,when the Alpha LISA detection system was evaluated with SEA simulated samples,the method showed good anti-interference ability to food matrix: in the simulated samples of SEA full-fat milk,the minimum detection limit(LOD)of Alpha LISA detection method was 0.1ng/m L.In the simulated samples of SEA dairy products with a concentration of 10%(w/v),the LOD of Alpha LISA was0.1ng/m L.In the simulated samples of SEA dairy products with a concentration of 20%(w/v),LOD can only reach 1ng/m L.In simulated samples of SEA soybean products and preserved meat at 10%(w/v)and 20%(w/v)concentrations,the LOD of Alpha LISA detection method can reach 0.1ng/m L.The LOD of the simulated samples of SEA peanut butter with a concentration of 20%(w/v)was 0.2ng/m L,and the LOD of the simulated samples of peanut butter,broad bean paste and ham sausage were all0.1ng/m L at different dilution mass fractions.When the Alpha LISA detection system was evaluated by the simulated plasma samples of botulinum toxin type A,the LOD of Bo NT-A in the plasma stock could only reach0.4ng/m L due to the influence of some components in the plasma,but the LOD of Bo NT-A in the buffer equal-ratio diluted plasma was 0.1ng/m L by Alpha LISA.In the simulated sample of botulinum toxin type A distilled water,the LOD of Bo NT-A detected by Alpha LISA could reach 0.1ng/m L.In the second part of this study,when the Alpha LISA detection system was evaluated with simulated t-2 toxin samples,the LOD of Alpha LISA detection method could reach0.05ng/m L in simulated T-2 toxin cornmeal samples with concentrations of 2%(w/v),5%(w/v)and 10%(w/v),which was consistent with that of the buffer solution.When the Alpha LISA detection system was evaluated with commercial simulated samples of T-2 toxin,the recovery rate of simulated samples of T-2 toxin at 2%(w/v)concentration was 89.49%~96.29%,and the recovery rate of commercial ELISA kit was80.25%~121.29%.The recovery rate of Alpha LISA detection method was significantly better than that of ELISA.When the concentration of simulated samples of T-2 toxin was diluted to 1%(w/v),the recovery rate of Alpha LISA was 87.16%~92.50%,and that of commercial ELISA kit was 37.52%~95.16%,indicating that the Alpha LISA method could still achieve a good recovery rate in simulated samples with low concentration.In this study,the Alpha LISA detection system of 11 toxins that could cause human poisoning was established.Compared with ELISA,this method could provide a rapid,highly specific and highly sensitive detection,which can provide reference for the detection of 11 toxins.