Preparation Of Monoclonal Antibody Against 2-Methyl-4-Chlorophenoxyacetic Acid And Its Development Of Immunochromatographic Strip

2-methyl-4-chlorophenoxyacetic acid（MCPA）was a widely used hormonal endogenous phenoxycarboxylic acid herbicide.It remains in the soil for a long time and usually causes serious pollution to subsequent crops and surface water,as well as certain toxic effects on human health.At present,the analysis and detection methods for 2-methyl-4-chloride residues in food are all based on chromatography technology,which has a long detection cycle,high equipment requirements,and cumbersome operation.This study synthesized 2-methyl-4-chlorohapten derivatives through the chemical modification pathway of increasing the connecting arm,and prepared 2-methyl-4-chlorohapten by coupling the carrier protein with carbodiimide method;On this basis,monoclonal antibodies targeting 2-methyl-4-chloride were prepared based on hybridoma technology and used as key components to study the immunochromatographic test strips of 2-methyl-4-chloride.The main research results are as follows:（1）This study synthesized three 2-methyl-4-chloro hapten derivatives M1,M2,and M3 with different connecting arm structures based on different pathways.They were characterized using quadrupole time-of-flight mass spectrometry.The results showed that the molecular weights of hapten M1 and M3 were 283.9946 and226.9811,respectively,which were consistent with the designed hapten derivatives.Meanwhile,the hapten M2 was determined using Fourier transform infrared absorption spectroscopy,The infrared absorption characteristic wavelengths were found to be 1750-1735 CM^-1and 3300-2500 CM^-1,indicating the successful introduction of C=O and O-H bonds.On this basis,three types of all antigens based on 2-methyl-4-chloride were prepared using the EDC/NHS method and characterized using a full wavelength ultraviolet spectrophotometer.All three types of all antigens had strong absorption peaks at 280 nm and retained specific absorption peaks of their hapten derivatives,indicating the successful synthesis and construction of all antigens,providing a basis for the later preparation of single gram antibodies and the establishment of immune analysis methods.（2）In the study of monoclonal antibody preparation,M1-BSA was used as an immunogen and hybridoma technology was used to obtain 10 strains of anti 2-methyl-4-chloride monoclonal antibodies,named 5B9,5B11,5D3,5G9,10E1,5F8,10C7,5D8,5D11,and 10F8,respectively.After antibody subtype identification,the subtypes of 5F8,10C7,5D8,and 5D11 antibodies are Ig G2b,the subtypes of 5B9,5B11,5D3,5G9,10E1 and 10F8 antibodies are Ig G1,and the light chain types of all10 antibodies are Ig?.Antibody gene sequencing was performed on 10E1 and 10F8antibody hybridoma cell lines,and the amino acid sequence and structural information of the antibodies were obtained.M2-OVA was used as the detection antigen,and the inhibition rates of 2-methyl-4-chloride antigen（standard concentration 100 ng/m L）based on antibodies 10E1,5B11,5D3,and 10F8 were 85%,84%,82%,and 81%,respectively.A 2-methyl-4-chloride competitive inhibition standard curve was established based on 10 monoclonal antibodies obtained.The results showed that the IC₅₀values ranged from 19.9 to 97.24 ng/m L,with 10E1 and 5F8 antibodies exhibiting the highest sensitivity,with IC₅₀values of 19.9 ng/m L and a linear detection range of 3.9-125 ng/m L.After cross reaction rate measurement,10antibodies showed ideal blocking effects on 2-methyl-4-chloride and its homologues2,4-D,2,4-D butyric acid.Among them,10E1,5B11,5G9,5B9 and 5D8 had a cross reaction rate greater than 100%based on 2,4-D,5B9 had a cross reaction rate of263.51%against 2,4-D butyric acid,and 5G9 had a cross reaction rate of 268.11%against 2,4-D,indicating that the obtained antibody is a broad-spectrum antibody targeting 2-methyl-4-chloride structural analogues.（3）In the study of colloidal gold immunochromatographic test strips,colloidal gold particles were prepared using the trisodium citrate reduction method and characterized by UV absorption spectroscopy.The results showed that the absorption peak was at 518 nm,which is consistent with the characteristic absorption peak of the target particle size of colloidal gold.On this basis,it is coupled with antibodies through gold sulfur bonds.After optimizing the coupling conditions,the optimal volume of 0.1 M K₂CO₃is 3μL.The optimal amount of antibody input is 6μg.After optimizing the detection conditions of colloidal gold immunochromatographic test strips,the T-line coating concentration was 2.5 mg/m L and the probe addition was 2μL.The results showed that the colloidal gold immunochromatographic test strip established in this study can detect 2-methyl-4-chloride in barley,with a minimum detection limit（v LOD）value of 78 ng/m L and a cutoff value of 313 ng/m L.Based on this detection system,the recovery range of 2-methyl-4-chloride in barley added samples was 82.2-101.1%,providing a preliminary work foundation for the rapid detection of 2-methyl-4-chloride in food in the later stage.