The Construction Of Ampicillin Single-chain Antibody And The Establishment Of Indirect Competitive ELISA Method

In order to improve the production level of animal husbandry,veterinary drugs have been used as a basic substance in animal husbandry,at the same time,they have also caused great harm to people's bodies.Therefore,the issue of veterinary drug residues has become a research hotspot.The most commonly used method for veterinary drug residue detection is immunoassay.The most influential factor for immunoassay is antibody.The stability of antibody plays a key role in the final result.This paper successfully obtained the single chain fragment variable of ampicillin by adopting genetic engineering technology.Based on this,an indirect competitive ELISA method was established to provide an economical,sensitive and rapid detection method for ampicillin residue detection.Total RNA was extracted from hybridoma cells of ampicillin antibody,and the first strand cDNA was synthesized by RT-PCR reverse transcription.The VH gene and the VL gene fragment were amplified by specific primers for the murine heavy chain variable region?VH?and light chain variable region?VL?genes,and after amplification,the VH gene fragment size was 340 bp.The amplified VL gene fragment was 320 bp in size,and the VH gene and VL gene were spliced into a single chain fragment variable gene?scFv?fragment by a flexible polypeptide linker?Gly4Ser?3 using overlap extension PCR?SOE-PCR?.The obtained scFv fragment is 750 bp in size,and then the single chain fragment variable gene?scFv?is subjected to Sfi I and Not I restriction endonuclease digestion,and the prokaryotic expression vector PLIP6/GN is also double-digested and double-digested.After the scFv and PLIP6/GN were ligated together,the recombinant plasmid was transferred into E.coli BL21,and the scFv was recombinantly expressed,cultured at 37°C overnight,and induced with 0.1 mmol/L IPTG,and cultured for 8 h to 12 h.An ampicillin-resistant single chain fragment variable-alkaline phosphatase fusion protein?AMP scFv-AP?was successfully obtained.Purification was carried out on a Nⁱ²+-NTA affinity chromatography column.Using the purified protein,a fast,simple and sensitive method for indirect competition of ampicillin was established by optimizing the indirect competitive ELISA conditions.The optimal detection conditions were as follows:the optimal working concentration of the coated antigen was0.25?g/In mL,the optimal single chain fragment variable dilution ratio is 1:32 000,and the antigen coating time is 37°C for 1 h.The blocking solution was 5%goat serum,the blocking time was 37°C for 2h,the antigen-antibody reaction time was 37°C for 1 h,Anti-His-tag antibody concentration is 1:4000and the coloration time was 15 min.Under the above conditions,the minimum detection limit?IC15?of the method was 0.83 ng/mL,the sensitivity?IC50?was 18.43 ng/mL,and the linear regression equation was y=-14.619x+68.623,R²=0.9887,The average recovery rate is 92.392%.Five kinds of veterinary drugs were selected for specific analysis.The results showed that the cross-reaction rates with amoxicillin and penicillin were 46.63%and 44.38%,respectively,and the cross-reaction rates with chloramphenicol,tetracycline and kanamycin were less than 0.01%.This study successfully prepared scFv antibody and established an indirect competitive EISA detection method by optimizing a series of conditions,which laid a foundation for ELISA rapid detection kit and had great significance in food safety detection.