Study On CLEIA And LFM-ICS Detection Techniques For T-2 Toxin And Zearalenone In Feed

T-2 toxin and zearalenone are widely found in nature and can pollute rice,wheat,oat corn,compound feed using these crops as raw materials and so on.Research showed that T-2 toxin and zearalenone are both one of the common highly toxic mycotoxins,which can cause human and animal toxicity injury,and harm human-health and prohibit the development of aquaculture industry.The study has synthetised complete antigen of T-2 toxin and zearalenone and then prepared of two monoclonal antibodies against T-2 toxin and zearalenone,Respectively,and then established chemiluminescence enzyme immunoassay(CLEIA)and lanthanide fluorescent microsphere immunochromatographic strip(LFM-ICS)to detect T-2 toxin and zearalenone in feed.The experiment contains the following four parts:Experiment I Preparation of two monoclonal antibodies against T-2 toxin and zearalenoneThe study has synthesized complete antigen T2-HS-JF-BSA and T2-HS-OVA,and then immunized BALB/c with T2-HS-JF-BSA.At length,the hybridoma technique was used to prepare monoclonal antibody against T-2 toxin.Hybridoma cells that can secrete anti-ZEN monoclonal antibody in the laboratory was resuscitatied and subcloned,and the positive sub-cell-strain that has been screened by indirect ELISA was extended culture and then inoculated BALB/c mice to prepare anti-ZEN McAb.The result of identifiction of immunological properties shows that two monoclonal antibodies have a high sensitivity and high specificity,and their IC50 are 0.36 ng/mL and 2.02 ng/mL,respectively.Experiment II Development of CLEIA for determining zearalenone in feedIndirect competitive chemiluminescence enzyme immunoassay(ic-CLEIA)and direct competitive chemiluminescence enzyme immunoassay(dc-CLEIA)were established for detection of zearalenone in feed and the two methods were compared.The optimum concentration of ZEN-CMO-BSA and dilution of monoclonal antibody(or McAb-HRP)were optimized by two factor-cross test to establish ic-CLEIA and dc-CLEIA.Under the optimized condition,the developed ic-CLEIA has a good linearity within 0.05-5.0 ng/mL and the calculated regression equation was y=-0.383 5x+0.417 9(R2=0.994 6).From regression equation,50%inhibitory concentration value was estimated as 0.611 ng/mL and average coefficient of variation of intra-and inter-assay are both less than 1.79%,and a limit of detection is 15 pg/mL.The developed dc-CLEIA has a good linearity within 0.025?5.0 ng/mL and the calculated regression equation was y=-0.386 5x+0.334 1(R2=0.991 8).From regression equation,50%inhibitory concentration value was estimated as 0.372 ng/mL and the average coefficient of variation of intra-and inter-assay are both less than<1.20%,and the limit of detection is 3 pg/mL The two method's recovery rates from fortified sample were 91.8%?112.1%and 87.3%?119.3%and CVs were less than 10.5%and 9.8%,respectively.Both methods are precise and sensitive,and can be used as an effective means for quantitative detection of ZEN.Compared with ic-CLEIA,dc-CLEIA is more suitable for the follow-up detecting study of ZEN in feed.Experiment III Development of CLEIA for determining T-2 toxin in feedIndirect competitive chemiluminescence enzyme immunoassay(ic-CLEIA)was established for detection of T-2 toxin in feed.The basis of the synthetic T2-HS-BSA and the prepared T-2 toxin monoclonal antibody,the optimum concentration of T2-HS-BSA and dilution of monoclonal antibody were optimized by two factor-cross test to establish ic-CLEIA.The results showed that the optimized concentration of T2-HS-BSA is 0.15?g/mL and the optimized dilution of McAb is 1:1536 000.Under the optimized condition,the developed ic-CLEIA has a good linearity within 25?300 pg/mL and the calculated regression equation was y=-0.641 1x+0.160 7(R2=0.998 3).From regression equation,50%inhibitory concentration value was estimated as 93.2 pg/mL and the average coefficient of variation of intra-and inter-assay are both less than 6.15%,and the limit of detection is 11.4 pg/mL.The results show that the developed CLEIA is accurate and has a high sensitivity,and can be used to deteermine T-2 toxin as an effective quantitative detection method.Experiment IV Development of LFM-ICS for determining zearalenone and T-2 toxin in feedIn study,the two McAb against ZEN and T-2 toxin were coupled with lanthanide fluorescent microspheres as the fluorescent probes,respectively.ZEN-CMO-BSA(or T2-HS-BSA)and goat anti-mouse IgG were sprayed into the NC membrane as the test line(T line)and control line(C line)to develop LFM-ICS for determining zearalenone or T-2 toxin in feed.Under the optimized condition,the developed LFM-ICS for determining zearalenone has a good linearity within 0.5-80 ng/mL and the calculated regression equation was y=-0.304 2x+0.619 6(R2=0.984).From regression equation,50%inhibitory concentrationvalue was estimated as 2.47 ng/mL and the average coefficient of variation of intra-and inter-assay are both less than 4.29%,and the limit of detection(LOD)and qualitative detection(QLOD)are 0.12 ng/mL and 1 ng/mL,respectively.The developed LFM-ICS for determining T-2 toxin has a good linearity within 0.125?2.0 ng/mL and the calculated regression equation was y=-0.609 7x+0.314 4(R2=0.990 6).From regression equation,50%inhibitory concentration value was estimated as 0.518 ng/mL and average coefficient of variation of intra-and inter-assay are both less than 4.36%,and the limit of detection(LOD)and qualitative detection(QLOD)are 0.109 ng/mL and 0.25 ng/mL,respectively.The results show that the developed LFM-ICS are precise and sensitive,have good repeatability and stability,which are suitable for the POCT detection of ZEN and T-2 toxin,and have a hopeful application prospect.