Preparation Of Bispecific Monoclonal Antibody Against Ochratoxin A And Aflatoxin B₁ And Its Application In Fluorescence Lateral Flow Immunoassay

Mycotoxins are toxic secondary metabolites produced by filamentous fungi in suitable conditions,posing a great threat to human and animal health.Ochratoxin A（OTA）is a secondary metabolite produced by Aspergillus and Penicillium.OTA is hepatotoxic,immunotoxic,and carcinogenic,listed as a group 2B carcinogen by the International Agency for Research on Cancer（IARC）.Aflatoxin B₁（AFB₁）is a secondary metabolite produced by Aspergillus parasiticus and Aspergillus flavus.AFB₁is the most toxic and carcinogenic substance among aflatoxins,grouped as a group 1carcinogen by IARC.As two types of highly toxic mycotoxins,OTA and AFB₁ widely exist in various crops including soybean,corn,and rice.More importantly,OTA and AFB₁ may coexist in a crop,which could cause increased toxicity by synergistic effects.Therefore,it is urgent to establish a method for simultaneous detection of OTA and AFB₁ in soybean,corn,and rice.In this study,a quantum dot beads（QBs）lateral flow immunoassay（LFIA）based on the bispecific monoclonal antibody（BsMAb）was established for simultaneous detection of OTA and AFB₁ in soybean,corn,and rice.The main research methods and conclusions are as follows:（1）Hypoxanthine guanine phosphoribosyl transferase deficient OTA hybridoma cells and thymidine kinase deficient AFB₁ hybridoma cells were prepared based on the8-azaguanine and 5-bromouracil deoxyriboside,respectively.And then the two kinds of deficient hybridoma cells were fused by using hybrid-hybridoma technology.Four kinds of BsMAb cell lines（2E7、2F7、4B2,and 3G9）that could specifically recognize OTA and AFB₁ were obtained after four rounds of subcloning.Indirect competitive enzyme-linked immunosorbent assay was used to evaluate the obtained BsMAb.The half maximal inhibitory concentrations(IC₅₀)for OTA were 0.65、0.58、0.60,and 0.67ng/m L,respectively,and the IC₅₀ for AFB₁ were 0.29、0.22、0.25,and 0.26 ng/m L,respectively.Besides,BsMAb had good specificity.The subtype of BsMAb with highest sensitivity was evaluated.The result indicated that BsMAb was Ig G1 subclass.（2）QBs-LFIA based on traditional anti-OTA monoclonal antibody（mAb）and anti-AFB₁ mAb was established for the detection of OTA and AFB₁ in soybean,corn,and rice,respectively.The coupling p H of mAb,the amount of EDC,the amount of mAb,the concentration of OTA-BSA,the concentration of AFB₁-BSA,and the volume of QBs-mAb probe were optimized.Under the optimum experimental conditions,the IC₅₀ of QBs-LFIA based on traditional anti-OTA mAb for detecting OTA in soybean,corn,and rice were 3.21、2.73,and 1.47μg/kg,respectively.The recovery rates were87.21%-132.49%and coefficients of variation were 0.51%-12.79%.The IC₅₀ of QBs-LFIA based on traditional anti-AFB₁ mAb for detecting AFB₁ in soybean,corn,and rice were 0.05、0.07,and 0.09μg/kg,respectively.The recovery rates were 80.38%-132.38%and coefficients of variation were 2.39%-11.63%.The detection limits of both methods meet the maximum limits of OTA and AFB₁ in soybean,corn,and rice.（3）OTA and AFB₁ may be present together in a crop.In order to improve detection efficiency,a fast and efficient QBs-LFIA based on the prepared BsMAb,which could specifically recognize OTA and AFB₁,was established for simultaneous detection of OTA and AFB₁ in soybean,corn,and rice.The coupling p H of BsMAb,the amount of EDC,the amount of BsMAb,the concentration of OTA-BSA,the concentration of AFB₁-BSA,and the volume of QBs-BsMAb probe were optimized.Under the optimum experimental conditions,the performance of BsMAb-based QBs-LFIA was analyzed.The sensitivity and specificity of this method were analyzed.The results showed that the IC₅₀ of BsMAb-based QBs-LFIA for detecting OTA and AFB₁ were 0.73μg/kg and0.01μg/kg in soybean,1.14μg/kg and 0.03μg/kg in corn,and 1.69μg/kg and 0.07μg/kg in rice.The BsMAb-based QBs-LFIA also had good specificity.The result of accelerated preservation experiment showed that this method was stable.The precision of the method was analyzed by recovery experiment.The recovery rates in soybean,corn,and rice were 80.20%-130.05%and coefficients of variation were 1.09%-10.41%.Therefore,the BsMAb-based QBs-LFIA has good specificity,stability,and accuracy,and it can be used for screening of OTA and AFB₁ in soybean,corn,and rice.To sum up,a BsMAb that could simultaneously identify OTA and AFB₁ was prepared in this study.Based on this novel BsMAb,QBs-LFIA that could simultaneously detect OTA and AFB₁ in crops（soybean,corn,and rice）was established,providing important technical support for the efficient quantitative detection of OTA and AFB₁.