Establishment And Development The Method Of Enzyme-linked Immunosorbent Asaay Kit Against Aflatoxins

Aflatoxins are a group of structurally similar mycotoxins produced by the fungi Aspergillus flavus and Aspergillus parasiticus,which are highly toxic with teratogenic,carcinogenic,mutagenic.AFB1,AFB2,AFG1 and AFG2 tend to contaminate fodders,foodstuffs and animal-derived foods,while the main food products susceptible to AFM1and AFM2 contamination are milk and dairy products.Governments in the world attach importance to aflatoxins‘contamination as a result that these contaminated fodders and foodstuffs can not only cause adverse health effects to animals and humans but are difficult to eliminate.In order to screen samples contaminated with aflatoxins,methods based on instruments and immunoassays have been established in recent years.Chromatographic assays have advantages with high sensitivity and great accuracy,wheras these methods require complicated sample pre-treatment,sophisticated equipment and skilled technicians,so there are some limitations for screening large amount of samples.Immunochemicanl methodes such as enzyme-linked immunosorbent assays（ELISA）are rapid,simple,high specificity and sestivity for aflatoxins determination in fodders,cereal and foodstuffs.Nowadays there are still some disadvantages of ELISA methods which have been used to screen aflatoxin residue.Therefore we aimed to create two different ELISA kits to detect aflatoxin residue,specific steps are as follows:1.The hapten AFB1-COM was synthesizd by using AFB1 and carboxymethoxylamine hemihydrochloride to create a reactive compound and identified successfully by LC/MS-IT-TOF.The antigen AFB1-EDC-OVA was synthesized successfully by coupling AFB1 with OVA,using EDC as a cross-linker.AFB1-EDC-OVA was identified by UV spectrum,result indicate it is success.2.Establishing and optimizing ELISA method by preparing AFB1 antibody through resuscitating cell,AFB1 as competitive drug and AFB1-EDC-OVA as antigen.Results showed that the optimum concentration of antigen was 1.08μg/m L and the best optimizing dilution of monoclonal antibody AFB1 was 1:1500;The standard curve regression equation was y=-0.6339x+1.5056,R²=0.9939,of which X=Lg（C（AFB1））,Y=B/B0.Furthermore,a good linearity was abtained at the range of 6.25～100 ng/L and the value of IC₅₀ was 38.58±1.95 ng/L（n=5）.Besides the inter-assay and intra-assay coefficient of variation were below 12.00%,while good seitivity of the mothed was8.61ng/L.The AFB1 antibody had specificity to AFB1,AFB2,AFG1 and AFG2,the cross-reactivity was 100%,7.29%,3.04%and 2.65%respectively,otherwise no cross-reactivity with other aflatoxins.3.The LODs and LOQs of the various samples（cow fodder,cattle fodder,chick fodder,adult chicken fodder,fattening pig fodder,maize as well as muscle,liver and kidney of pig,beef together with chicken）are 5.54-239.28 ng/L and 8.36-424.43 ng/L respectively.Four aflatoxin drugs（AFB1,AFB2,AFG1,AFG2）at three vary concentration（1×LOQ,2×LOQ,4×LOQ）were spiked into these different samples,then the spiked samples were screening by ic-ELISA of AFB1 method after samples preparation.Results showed that the recoveries of five kinds of fodders were between70.15%-111.33%,and inter-assay and intra-assay coefficient of variation was below13.50%;the recovery of maize was 74.55%-107.82%,and the inter-assay and intra-assay coefficient of variation was below 15.00%;the recovery of nine kinds of animal tissues was between 71.69%-110.27%,and the inter-assay and intra-assay coefficient of variation was below 14.00%.Therefore,the ELISA kit based on AFB1 antibody can be used to detecte four kinds of aflatoxins in 15 different samples and the method was accuracy and realiable.An excellent correlation（R²=0.9968）between ic-ELISA and HPLC-MS/MS results was observed,which suggested that the present ic-ELISA is reliable.4.Establishing and optimizing ELISA method by preparing AFM1 antibody through resuscitating cell,AFM1 as competitive drug and AFB1-EDC-OVA as antigen.Results showed that the optimum concentration of antigen was 4.0μg/m L and the best optimizing dilution of monoclonal antibody AFM1 was 1:2000;The standard curve regression equation was y=-0.3844x+1.3206,R²=0.9908,of which X=Lg（C（AFM1））,Y=B/B0.A good linearity was abtained at the range of 5～405 ng/L and the value of IC₅₀was 136.38±10.35 ng/L（n=5）.In addition,the inter-assay and intra-assay coefficient of variation were below 12.50%,while good seitivity of the mothed was 11.70 ng/L.The AFM1 antibody had no specificity to other aflatoxins.The LODs and LOQs of the samples（milk as well as muscle,liver and kidney of pig,beef together with chicken）are10.77-14.59 ng/L and 18.44-27.15 ng/L respectively.AFM1 at three vary concentration（1×LOQ,2×LOQ,4×LOQ）were spiked into these samples,the spiked samples were screening by ic-ELISA of AFM1 method after samples preparation.Results showed that the recoveries of five kinds of various samples were between 73.73%-106.16%,and the inter-assay and intra-assay coefficient of variation was below 15.00%.Hence,the ELISA kit based on AFM1 antibody can be used to detect AFM1 in 10 different samples and the method was accuracy and realiable.An excellent correlation（R²=0.9985）between ic-ELISA and HPLC-MS/MS results was observed,which suggested that the present ic-ELISA is reliable.5.It was necessary to assess the stability of antibodies and antigen,hence we stored antibodies of AFB1 antibody and AFM1 antibody,AFB1-EDC-OVA antigen at 37℃for accelerating sability experiments.Resuts indicated that no matter antibodies or antigen can be stored stablitily for one year under conventional condition.We created two ELISA kits to detecte aflatoxin residue rapidly through compounding hapten and antigen as well as preparing AFB1 antibody.On the one hand,ELISA kit based on AFB1 antibody can detecte aflatoxin（AFB1,AFB2,AFG1 and AFG2）residue in 5 kinds of fodders,mazine and 9 kinds of animal tissues.On the other hand,ELISA kit based on AFM1 antibody can detecte AFM1 residue in milk and 9 kinds of animal tissues.More impotantly,these two kits are accuracy and sensitivity.Meanwhile,stablitility of antigen and antibodies have been assessed.In constrat to the same ELISA kits of aflatoxins have published,kits we created have advatages with high sensitivity,compound of hapten,antigen simplely and less material as well as screening more samples,etc.All in all,these ELISA methods play an indispensable role not only in academics but also in application.