Preparation Of Dicamba Nanobodies And Its Immunoassay Method

Dicamba,also known as paraquat,is a highly effective and low toxicity herbicide with selective and systemic conductivity.In recent years dicamba has gained renewed momentum with the development of dual genetically modified soybeans and cotton tolerant to dicamba and glyphosate.However,dicamba has a high volatility and is highly susceptible to drift damage.Therefore,the development of efficient and sensitive dicamba detection techniques is essential for real-time monitoring of trace dicamba in the environment and evaluation of its drift damage.Nanobody-based enzyme immunoassay techniques are highly efficient,rapid,sensitive and immediate,and to some extent compensate for the inability of accurate and efficient large-scale instruments to be used for rapid detection in the field.However,various enzyme immunoassay-based methods for dicamba detection currently rely on polyclonal or monoclonal antibodies,and no nanobody-based detection of dicamba in the environment has been reported at home or abroad.Based on the above problems,this study was carried out with dicamba as the research object and the main research contents are as follows:1.In this study,dicamba polyclonal antibodies were purified from the serum after the fifth immunisation by immunising camels and collecting camel sera for potency evaluation,and an enzyme immunoassay method based on polyclonal antibodies for dicamba was established.The IC50 value of the method was 15.32 μg/mL,the linear range was 4.52-56.29 μg/mL,and the minimum detection limit was 4.52 μg/mL.2.The serum of camel after the fifth immunization was collected,RNA was extracted and reverse transcribed into cDNA,and the gene fragment of the nanobody(VHH)was obtained by two rounds of amplification,which was transformed by enzymatic ligation with the phage vector pComb3x,and a phage-displayed nanobody library was successfully constructed through the infestation of the auxotrophic phage M13KO7.On this basis,a nanobody specifically recognizing dicamba was successfully eluted by solid phase elution using both homologous and heterologous encapsulation.The IPTG induction concentration,induction time and induction temperature of this nanobody were also optimized to prepare a large number of dicamba nanobodies for prokaryotic expression.3.Nb-242 was selected as the optimal nanoantibody by indirect competitive enzyme immunoassay,and an enzyme-linked immunosorbent assay(ELISA)method based on Nb-242 was constructed.The IC50 value of the method was 0.93 μg/mL,and the linear range was 0.11～0.93 μg/mL with the lowest detection limit of 0.11 μg/mL.The highest cross-reactivity was observed with 2,3,6-trichlorobenzoic acid and almost no cross-reactivity was observed with other compounds.In addition,matrix effects were evaluated and spiked recoveries were validated for tap water and soil,with spiked recoveries ranging from 97%to 104%and 98%to 110%,respectively.4.To further improve the sensitivity of the assay,a FLEIA analytical method based on Nb-242 was constructed.The method showed an IC50 value of 22 ng/mL,a linear detection range of 2 to 229 ng/mL and a minimum detection limit of 2 ng/mL,which was 50-fold more sensitive compared to the ELISA assay.This indicates that the developed FLEIA assay based on dicamba nanobodies is more rapid and accurate in detecting actual environmental samples.5.To initially investigate the mechanism of specific recognition of dicamba by the nanobodies,homology modelling of Nb-242 was performed using YASARA,which was evaluated by ERRAT,Procheck and Verify3D to obtain a high-quality model,and the nanobodies were molecularly docked to dicamba small molecules,and the key binding sites were predicted to be Trp 82,Arg 69 and Arg 72.