Expression And Evaluation Of Genetically Engineered Antibodies To Thiacloprid And Ochratoxin A And Their Application In Food Detection

Pesticide residues and mycotoxin contamination are important hazards in the production,storage,processing,and marketing of agricultural products.Thiacloprid（TCL）is widely used in agricultural production due to its broad-spectrum and highly effective characteristics and has high residues.Ochratoxin A（OTA）,a typical mycotoxin,is a highly hazardous and widespread contaminant and has been found in cereals,fruit,and vegetables.However,little research has been published on the simultaneous detection of these two harmful substances,and therefore it is important to establish methods for the rapid detection of these two contaminants.Currently,most of the common rapid detection products are single-component tests based on traditional immuno-antibodies,which cannot detect pesticides and toxins simultaneously,and are complex and time-consuming to prepare.A bispecific antibody recognition element based on recombinant genetic engineering technology is a good approach to solving these problems and can be rapidly expressed in vitro to achieve simultaneous detection of harmful substances across classes.In this work,single-chain variable fragments（scFv）against thiacloprid were expressed in E.coli BL21 and P.pastoris GS115,and single-chain antibody against thiacloprid,full-length antibody（IgG）,bispecific antibodies（Bs Abs）to thiacloprid and ochratoxin A were expressed in mammalian cells Expi293F.The effects of different hosts and antibody forms on antibody production and activity were compared.Time-resolved fluorescent immunoassay strips based on IgG antibodies were prepared and a competitive ELISA assay for thiacloprid and ochratoxin A based on bispecific antibodies was developed.The main studies and conclusions were as follows:（1）Preparation of scFv and IgG antibodies to TCL based on different expression systems with a comparison of antibody yield and activity.The E.coli expression vector p ET-28a-scFv,the P.pastoris expression vector p PIC9K-scFv,the mammalian cell expression vector p CMV-scFv,the mammalian cell heavy chain expression vector p CMV-HC and the mammalian cell light chain expression vector p CMV-LC were designed.The plasmids were expressed in E.coli BL21,P.pastoris GS115,and mammalian cell Expi293F,respectively.The results showed that the yields of single-chain antibodies expressed in E.coli BL21,P.pastoris GS115,and mammalian cells Expi293F were 7,3.2,and 1.6 mg/L,respectively.scFv antibodies produced based on BL21 were almost inactive,scFv antibodies produced based on GS115 were poorly active,the scFv antibody expressed by Expi293F had activity,the IC₅₀ value of competitive ELISA standard curve was 14.87 ng/m L,the IgG antibody expressed by Expi293F had the best activity,and the yield was about 35 mg/L,the affinity constant was 1.2 x 10⁹ L/mol.The IC₅₀of ELISA standard curve based on IgG antibody was 2.63 ng/m L under the optimal conditions.By comparing the three expression and antibody forms on antibody yield and activity,it was concluded that IgG antibodies expressed in mammalian cells Expi293F had the highest yield and best activity.（2）Based on the previously constructed genetically engineered IgG antibody,the antigen was synthesized,and the time-resolved fluorescence immunoassay was constructed and applied in food.The fluorescent immunoassay strips for thiacloprid were developed with a linear range of 0.01-10 ng/m L,linear equation of y=-19.113log X+62.038,IC₅₀ of 4.268 ng/m L,and LOD value of 0.003 ng/m L.The intra-and inter-batch coefficients of variation were 0.40%and5.18%,respectively,with high sensitivity and stability.The cross-reactivity with the structural analog acetamiprid was 19.27%.Based on the molecular docking analysis,it was concluded that the cyanine group in the structures of acetamiprid and thiacloprid could form a hydrogen bond with the ILE-100 residue of the antibody.The recoveries of the method were 79.4%-118.6%when applied to fruits and vegetables,and 92.1%-107.6%by high performance liquid chromatography tandem mass spectrometry（HPLC-MS）,which showed good accuracy.（3）A bispecific antibody expression vector was constructed to establish an ELISA assay for the simultaneous detection of thiacloprid and ochratoxin A.The E.coli BL21 expression vector p ET-28a-OTA and mammalian cell Expi293F expression vector pc DNA3.1-OTA-TCL were designed and expressed in BL21 and Expi293F,respectively,and the recognition between ochratoxin A,thiacloprid and bispecific antibodies were investigated by molecular docking.The results revealed that the expressed OTA nanobody yield was about 8.6 mg/L with an affinity constant of 2.3 x 10⁵ L/mol and an IC₅₀ of 22.65 ng/m L for the ELISA standard curve.The bispecific antibody yield was about 2 mg/L,the linear range for the detection of thiacloprid was0.05-1000 ng/m L.The linear range was 0.05-500 ng/m L with an IC₅₀ of 16.69 ng/m L and a LOD of 0.788 ng/m L for the detection of ochratoxin A.The spiked recoveries for the detection of TCL and OTA in grape samples were 77.9%-125.5%and 76.2%-87.0%,and the recoveries of TCL and OTA by HPLC-MS were 90.1%-107.3%and 84.5%-94.3%,respectively,the accuracy of the method was good.In conclusion,in this study,the effects of different hosts and antibody forms on antibody yield and activity were comparatively analyzed by three expression systems,and thiacloprid scFv,IgG antibody,and bispecific antibody were obtained.We also constructed rapid detection test strips and simultaneous detection kits to achieve simultaneous detection of cross-category contaminants.