Construction Of Nanobody-based Enzyme Immunosensing Method For Ochratoxin A And Antibody Directed Evolution

Ochratoxin A?OTA?,one of the most harmful mycotoxins,is a toxic metabolite mainly produced by fungi such as Aspergillus and Penicillium.It has strong toxic effects on humans or animals,such as nephrotoxicity,liver toxicity,neurotoxicity,teratogenicity,and carcinogenicity.Therefore,it is of great significance to establish a highly sensitive and efficient detection method for OTA.This study focuses on improving the sensitivity of the detection method from two directions,including the development of an efficient enzymatic immunosensor method for OTA and affinity improvement of the anti-OTA nanobodies.The main research results are shown as follows:1. Calcein can combine with Ce³⁺to form a complex to quench its fluorescence.The formation of Ce PO₄ can make calcein dissociate from the complex and its fluorescence is renatured.Based on the principle that calcein and PO₄^3-can competitively combine with Ce³⁺,a phosphate-triggered fluorescent immunosensor system was constructed.After using the optimal working conditions,the Nb-AP mediated PT-FIA has a half maximal inhibition concentration(IC₅₀)of 0.46 ng/m L,a limit of detection(IC₁₀)of 0.12 ng/m L,and a linear range(IC_20-80)of 0.2-1.26 ng/m L,respectively.The recovery experiment indicated acceptable accuracy and precision of the Nb-AP-mediated phosphate-triggered fluorescence immunoassay,and the results were validated by high performance liquid chromatography with fluorescence detector.Thus this proposed method is applicable to sensitive,rapid,and low-cost detection of OTA and other toxic analytes with low molecular weight in food and environment.2. Nanobody?Nb?,the smallest genetically engineered antibody,has the complete antigen-binding properties of monoclonal antibodies.Due to its structural advantages, such as simplicity,stability,easy modification,easy realization of soluble expression, it can be used for affinity maturation of antibodies in vitro.In this study,the anti-OTA nanobody Nb28 was used as a model,and its three-dimensional structure model was obtained by SWISS-MODEL.After minimizing the energy,it was used to dock with OTA using Autodock4.2 and the key amino acid sites of the Nb28 for interacting with OTA were predicted.Finally,the four key amino acid sites were further determined after alanine scanning and ELISA,namely Gly53,Met79,Ser102,and Leu149.3. After homology modeling,molecular docking and alanine scanning verification,Gly53,Met79,Ser102,and Leu149 were determined as the key amino acids of Nb28 for interacting with OTA.Two site-directed saturated mutation libraries were constructed by two-site mutation against those four key amino acids.After biopanning and identification,a mutant Nb-G53Q&S102D was obtained with a half maximal inhibition concentration(IC₅₀)of 0.29 ng/m L and a KD value of 52 n M,which is1.4-fold and 1.36-fold lower than that of the original sd Ab,respectively.The computer simulation analysis indicated that the hydrogen bond,hydrophobic interaction,and side chain steric hindrance of amino acid residues are critical for the binding affinity of nanobodies.Overall,this study provides a reference model of improving the sd Ab's affinity to the low-molecular-weight compound.