Preparation Of Monoclonal Antibodies Against Colistin Sulfate And Development Of An ELISA Method

Colistin sulfate(CLS),also known as polymyxin E or colistin,is a polypeptide antibiotic produced by Paenibacillus polymyxa,mixed by CLS-A and CLS-B.It is mainly used for the treatment of sensitive bacteria in clinical veterinary and has been widely used as growth promoter in animal feed.Because of the toxicity of CLS such as nephrotoxicity and neurotoxicity,it is very easy to cause serious health impacts when people ingest foods with excessive CLS residues for a long time.At present,China's Ministry of Agriculture has banned CLS as an animal growth promoter in animal husbandry.In order to prevent the abuse and illegal addition of CLS,effective detection methods should be used to monitor the residues of CLS in animal food.At present,the main methods for the determination of CLS residues mainly include microbiological method,liquid chromatography(LC),liquid chromatography tandem mass spectrometry(LC-MS/MS),gas chromatography tandem mass spectrometry(GC-MS/MS),capillary chromatography,etc.However,the instruments and equipments required for the above several methods are relatively expensive.Besides the inspectors must have professional experimental techniques to avoid the impact on the efficiency and accuracy of the sample detection and to ensure the correctness of sample detection.Therefore,it is urgent to establish a rapid,simple and sensitive method for CLS detection.Based on CLS artificial antigen,this experiment established an ELISA method for detection of CLS residues in animal-derived foods by using monoclonal antibody preparation technology and enzyme-linked immunosorbent assay.The method is simple in operation,but has high specificity and high sensitivity,which is suitable for rapid detection of large quantities of samples in animal husbandry and animal product processing.1?Synthesis and identification of CLS complete antigensCLS is a small molecule polypeptide antibiotic which belongs to hapten and does not possess immunogenicity.CLS has active amino groups in its molecular structure,so this experiment used a heterobifimctional cross-linking agent 3-Maieimidobenzoicacid-N-hydroxy- succinimide ester(MBS)to couple amino groups of CLS molecules to thiol groups of bovine serum albumin(BSA)introduced by thiol exchange reduction of its disulfide bonds with dithiothreitol and forming a spacer arm between CLS molecules and carrier proteins to synthesize the immune antigen.The amino groups of CLS were coupled with the amino groups of carrier protein ovalbumin by a two-step glutaraldehyde method to synthesize the coating antigen.The synthesized complete antigens were identified by SDS-PAGE,Ultraviolet Scanning and immunological methods.And the results showed that CLS was successfully coupled with carrier proteins.The protein concentrations of CLS-BSA and CLS-OVA were 1.8 mg/mL and 4.6 mg/mL respectively by BCA protein assay kit.2.Preparation of monoclonal antibody against CLSUsing CLS-BSA as immunogen to immunize BALB/c mice with routine immunization procedures and the serum titer of immunized mice was determined to be 1:12800 or more after five immunizations.The spleen cells of immunized mice were fused with myeloma cells by using hybridoma cell technology,and three hybridoma cell lines—3 A2,5E2,9G7—which could stably secreting monoclonal antibodies against CLS were obtained by ELISA assay and gradient dilution methods.Ascites were prepared by in vivo induction in mice,which were 7 mL?14.7 mL,12.4 mL and the protein concentrations were 14.32 mg/mL?32.57 mg/mL and 21.8 mg/mL,respectively.The subtype of the antibodies were identified as IgM and the light chain were kappa chain and the titer of the antibodies were 1:12800 by ELISA.The antibodies had good specificity and no cross-reaction with Bacitracin,Monensin sodium and Vancomycin hydrochloride.3.The establishment of CiELISA detection methodThe 5E2 ascites were used to establish a square matrix ELISA test to determine the optimal working concentration of the detection antigen was 1:5000,the working concentration of the antibody was 1:3200,and the optimal blocking solution was 0.5%gelatin,thereby establishing an indirect competitive ELISA method for detecting CLS.When CLS concentration was in the range of 10?1000 ng/mL had a good linear relationship and the equation was y=0.4441x-0.3691(R2=0.9925),its IC50 was 89.57 ng/mL,LOD and LOQ were 9.66 ng/mL,25.71 ng/mL.4.Addition and recovery of CLS in porkThe addition and recovery of CLS used the pork sample as a substrate.The 8-fold dilution of the treated sample could substantially eliminate the interference of the sample matrix.When the CLS concentration was in the range of 20?1000 ng/mL,the linear relationship was good and the equation was y=0.4661x-0.4432(R2=0.9903).When adding 50,500 and 1000 ng/mL CLS to the pork,the recovery was 78.66?104.02%and the coefficient of variation was 3.25?6.86%.