Preparation Of Monoclonal Antibodies Against Chloramphenicol And 2-methylisoborneol And Establishment Of Enzyme-linked Immunosorbent Assays

Chloramphenicol(CAP)is a highly effective broad-spectrum antibiotic,which can inhibit the growth of bacteria by hindering the synthesis of bacterial proteins.Due to its low price and stable antibacterial properties,it has been widely used in animal husbandry and aquaculture.However,Researches have shown that CAP can easily accumulate residues in humans and animals,and has strong toxic and side effects.At present,CAP has been prohibited from using in animal and fish feed in the China,European Union,etc.However,under the drive of economic interests,the illegal use of CAP in animal and aquaculture still occurs from time to time.2-Methylisobomeol(MIB)is an odor substance that is easily enriched in fish,shrimp and other aquatic products,seriously affecting the taste and quality of aquatic products,resulting in greater economic losses.In order to ensure the safety of animal foods and improve the quality of aquatic products,it is urgent to establish high sensitivity and selectivity analysis methods for the determination of CAP in animal feed and animal foods and MIB in aquatic products.The first part of the study was to prepare a specific monoclonal antibody(mAb)against CAP by hybridoma antibody preparation technology,then based on mAb to establish a highly sensitive and specific enzyme-linked immunosorbent assay(ELISA)for CAP.First,three immunogens were prepared and used to immunize mice.Splenocytes from high titer mice were selected for cell fusion.Then,two lines of hybridoma cells(1B1 and 2D2)secreting specific antibodies were successfully screened by culturing and screening hybridoma cells.Under optimal conditions,the values of the 50%inhibition concentration(IC50)and limit of detection(LOD)of the ELISA in phosphate-buffered saline(PBS)based on 2D2 mAb for the detection of CAP were 0.46 ng mL-1 and 0.06 ng mL-1,respectively.There was no cross-reactivity(CR)of the ELISA with other antibiotics(florfenicol,thiamphenicol,etc.).Shrimp,feed and milk samples were spiked with different content of CAP and detected by ELISA after extraction and dilution.The recoveries and coefficients of variation(CV)of the ELISA for CAP from three spiked samples were 80.6-116.0%and 5.5-15.4%(n=3),96.2-111.0%and 7.0-13.5%(n=3),and 79.7-109.8%and 5.8-13.3%(n=3),respectively.In addition,the spiked samples were pretreated with solid phase extraction(SPE)and detected by liquid chromatography tandem mass spectrometry(LC-MS/MS).Although ELISA(detection results are expressed in terms of recovery)has a good correlation with LC-MS/MS-SPE,the results of LC-MS/MS-SPE are significantly lower than those of ELISA because of the low efficiency of SPE extraction.Thus,we established an immunoaffinity clean-up(IAC)for the pretreatment of CAP spiked samples by covalently immobilizing CAP mAb on the suface of CNBr-activated Sepharose-4B gel.Under optimized conditions,the mAb-fixed gel was mixed with the extract of spiked sample to extract and purify the target.IAC technology is easy to operate and the extraction efficiency is significantly improved.The recoveries of CAP from spiked samples measured by LC-MS/MS-IAC were 78.6-106.7%with the CV lesser than 11.1%(n=3).The second part of the study was to synthesize the immunogen of MIB by chemically modifying the analog of MIB(camphor)and to prepare MIB mAb.There is only one active group(hydroxyl)in the structure of MIB,and the pure product is expensive.By reacting with a Grignard reagent of allyl bromide,the camphor was introduced with an allyl functional group,followed by oxidation of the double bond,and a derivative highly similar to the MIB structure was successfully synthesized.Then,the immunogen and the coated antigen were prepared by linking activated derivative to the carrier protein.Finally,two lines of hybridoma cells(3A4 and 6C2)were successfully screened by hybridoma antibody preparation technology,and monoclonal antibodies will be prepared in large quantities.