Preparation Of Genetically Engineered Antibody Specific For Chloramphenicol And Development Of Immune Detection Method

The extensive use of the various veterinary drugs poses a threat to human health,although it has improved the yield of animal foods.The animal food exports of our country are also affected because of residues of veterinary drug.Immunoassay is a commonly used method for detection of veterinary drug residues.Antibody is the key reagent of immunoassay and its stability will greatly influcence the accuracy of immune detections.In this study,genetically engineered antibody specific for chloramphenicol was obtained using molecular techniques and an immune detection method was established with this antibody.This work provides an effective and stable method for chloramphenicol detection.Total RNA was extracted from a hybridoma clone 10A3B6E,which secrets monoclonal antibody against chloramphenicol,then cDNA was obtained after reverse transcription.The cDNA and degenerate primers were used to performe PCR amplification and the heavy chain variable region fragments(VH)and the light chain variable region fragments(VL)were obtained.The VH and VL were inserted into pMD18-T vector for a large scale sequencing.Meanwhile,the monoclonal antibody was digested using papain to generate the Fab.The light chain and heavy chain of Fab were separated using SDS-PAGE method and applied to Orbitrap mass spectrometry analysis for obtaining the amino acid sequence fragments of the antibody.By comparison of the results of DNA sequencing and the resulst of mass spectrometry analysis,the VH and VL specific for chloramphenicol were attained.The VH and VL were 341 bp and 324 bp in length,respectively.The VH,VL and a Linker sequence were linked to form a single chain variable fragment(scFv)with a length of 711 bp using Overlap Extension PCR method.The scFv fragment was digested by Sfi Ⅰ and Not I and linked with pLIP6/GN vector,which was also digested by the two enzymes.The recombinant vector was transformed into BL21(DE3)strain for heterogeneous expression of the scFv.CAP scFv-alkaline phosphatase(CAP scFv-AP)fusion protein was synthesized after induced by 0.1 mmol/L IPTG at 25℃,180 rpm incubator for 10-14 h.The protein was seperated and purified by Ni2+-NTA affinity chromatographic column.After optimization of the detailed conditions,an simple,fast and practical direct competitive ELISA method was developed using the purified protein as key reagent.The optimized conditions for the competitive ELISA were listed as follows:coating antigen at 0.1 μg/well,blocking with 1%skim milk for 1 h,diluting sampls using 0.02 mol/L TBS(pH 5.7).The IC50 and IC15 of this method reached 6.92±0.24 ng/mL and 1.11±0.05 ng/mL,respectively.Three structural analogs of chloramphenicol and 2 veterinary drugs were selected for specificity analysis.Cross-reactivities of this method to chloramphenicol sodium succinate,thiamphenicol,florfenicol,tetracycline and streptomycin were 42.34%,0.37%,0.22%,0.22%and 0.12%,respectively,which indicated the specificity of the developed ELISA was high.Cod and shrimp were selected as real samples and added 10,30,100 ng/g chloramphenicol to them,respectively.The recovery rates were 72.53%-107.67%and the coefficient of variation were below 10%,which indicated that the method had great accuracy.The current study provides an effective preliminary screening method for chloramphenicol detection in large amount of samples.It is helpful to improve the safety of animal foodstuff.