Development Of Anti-OTA And Anti-AFB₁ Nanobody Test Strips And Detection Of OTA Based On FRET System

Aflatoxin B1(AFB1)and ochratoxin A(OTA),as toxic and harmful small molecule substances widely distributed in food and feed,seriously threaten the health of humans and animals,mainly manifested as liver and kidney damage and teratogenic carcinogenesis.Because of its small size,high stability,good water solubility,and easy modification of genetic information,Nanobodies have been widely used in the field of food safety as target tracers.The simple,economical and fast immunochromatographic test strips are widely used in the detection of mycotoxins,and the simultaneous detection of multiple toxins and the detection of high sensitivity have become the problems we need to solve.Homogeneous FRET technology detects energy targets by controlling the effective spectral overlap,spatial distance,and dipole orientation between the fluorescent donor and the fluorescent acceptor,and using fluorescent signals as the signal output.More conventional detection methods have higher sensitivity,so choosing the right donor and acceptor,and establishing the best FRET system have become the key to solving the problem.In this study,a dual detection strip for AFB1 and OTA was developed by expressing and purifying Nanobodies against AFBi and OTA.A recombinant expression vector was constructed to express fusion protein of anti OTA nanobody with YFP specificity as a fluorescent donor,which was conjugated to a target competitor.Nano-gold was used as a fluorescent acceptor to establish a FRET system.The specific research is as follows:1.Development of dual immunochromatographic test strip for simultaneous detectionof OTA and AFB1The recombinant expression vectors pET-CTB-VHH28 and pET-CTB-G8 were transferred into Escherichia coli for expression,pentavalent CTB-VHH28 was obtained with a molecular weight of about 150 KDa.The sensitivity of the protein was detected by indirect competition ELISA to measure protein activity,and the IC50 was 0.68 ng/mL.The obtained monovalent nanobody CTB-G8 has a molecular weight of about 30 KDa.The sensitivity of the protein was detected by indirect competition ELISA to measure protein activity,and the IC50 was 1.97 ng/mL.AuNFs with a particle size of 56 nm were prepared.A dual test strip was prepared by fixing OTA-BSA and AFB1-BSA and Anti-His-mAb on the NC membrane to detect OTA and AFB1.Optimized the coupling of CTB-G8 and Anti-CTB-mAb with AuNFs to obtain the optimal pH,the optimal ratio of Anti-CTB-mAb and CTB-VHH28,the optimal amount of CTB-G8,and the gold probe AuNFs@Anti-CTB mAb@CTB-VHH28 and AuNFs@CTB-G8 are optimally added,and OTA-BSA and AFB1-BSA are optimally fixed.IC50 for OTA is 0.378 ng/mL,LOD is 0.0957 ng/mL,IC50 for AFB1 is 1.541 ng/mL,and LOD is 0.184 ng/mL.Nanobodies were prepared to replace traditional monoclonal antibodies,as well as flower-shaped nano-gold with bumps and huge specific surface area,which achieved high sensitivity detection of AFB1 and OTA toxins.2.Establishment of FRET system based on anti-OTA specific fluorescent nanobodiesand AuNSsThe fluorescent protein VHH28-YFP was expressed as a fluorescent donor,and MNPs were combined to prepare energy acceptor probes MNPs@Cys@AuNPs@OTA-BSA.The optimal coupling amount of Cys,the amount of AuNSs added,and the optimal coupling amount of OTA-BSA were optimized VHH28-YFP optimal input.By calculating the quenching efficiency E,a standard curve for the FRET system was established,and the LOD was 8.05 pg/mL.The results show that homogeneous FRET has higher sensitivity than conventional detection methods.