Development Of Indirect Competition ELISA Kit For AFB₁,T-2 Toxin And DON

T-2 toxin,AFB1 and DON are all toxic natural pollutants produced by fungi,and are common mycotoxins in feed materials and feeds.The pollution of feed materials and feeds.is common.It is generally mentioned in the literature that the detection shows a high positive rate.Among them,AFB1 is the most toxic,teratogenic,carcinogenic and mutagenic,and most often causes liver cancer;T-2 toxin can cause various reactions such as bleeding and immune system disorders;DON can cause vomiting,immune stress,antifeeding and other discomforts.reaction.All three toxins can cause damage to animals and people,and ultimately lead to economic loss or health problems.Therefore,the rapid and sensitive detection method is more suitable for the current detection needs.The immunological detection method is simple in operation and short in use,just meeting the above conditions.In this experiment,ic-ELISA kits for three toxins were developed by monoclonal antibodies with high sensitivity and specificity.Test Ⅰ Preparation of T-2 Toxin Monoclonal AntibodyFemale Balb/c mice were immunized with the existing T-2-OVA complete antigen in our laboratory,and specific polyclonal antiserum was produced.The titer was 2.5×105 and the IC50 was 10.98 ng/mL.The spleen cells were fused with SP2/0 cells,screened and subcloned.Two cell lines were obtained,which were 10A6 and 10D10 respectively.The 10A6 with better inhibition effect was prepared and purified by ascites.The titer was 5.12×105.The IC50 is 1.63 ng/mL,and there is no cross-reaction with AFB1,OTA,DON,and ZEA.The CR is less than 0.01,and there is a slight cross-reaction with HT-2,which is highly specific.Test Ⅱ Preparation of T-2 ic-ELISA kitBy using the prepared T-2 toxin monoclonal antibody,after optimization of the working concentration of the coating and the monoclonal antibody,reaction time,blocking solution and blocking time,combined with the relevant experience of the laboratory preparation kit,the production was successfully targeted.TC-ELISA kit for T-2 toxin detection.The detection limit of the kit for feed samples is 0.0611 ng/mL,the detection range is 0.1230 ng/mL-8.1759 ng/mL,the recoveries of standard addition are between 97.7%-106.2%,and the intra-assay coefficient of variation is less than 10%.The coefficient of variation between the two is less than 15%.After paired t-test,the difference from HPLC results is not significant,and it can be applied to the actual detection of large samples.Test Ⅲ Preparation of AFB1 ic-ELISA kitBy using the existing AFB1 monoclonal antibody in the laboratory,after optimization of the working concentration of the coating and the monoclonal antibody,reaction time,blocking solution and blocking time,combined with the relevant experience of the laboratory preparation kit,the production was successful.ic-ELISA kit for AFB1 toxin detection.The detection limit of the kit for feed samples is 0.209 ng/mL,the detection range is 0.342 ng/mL-6.597 ng/mL,the recoveries of standard addition are between 93.7%-107.6%,and the intra-assay coefficient of variation is less than 10%.The coefficient of variation between the two is less than 15%.After paired t-test,the difference from HPLC results is not significant,and it can be applied to the actual detection of large samples.Test Ⅳ Preparation of DON ic-ELISA kitBy using the existing DON monoclonal antibody in the laboratory,after optimization of the working concentration of the coating and the monoclonal antibody,reaction time,blocking solution and blocking time,combined with the relevant experience of the laboratory preparation kit,the production was successful.ic-ELISA kit for DON toxin detection.The detection limit of the kit for feed samples is 10.32 ng/mL,the detection range is 17.017 ng/mL-320.539 ng/mL,the recoveries of standard addition are between 97.7%-106.2%,and the intra-assay coefficient of variation is less than 10%.The coefficient of variation between the two is less than 15%.After paired t-test,the difference from HPLC results is not significant,and it can be applied to the actual detection of large samples.