Establishment And Application Of Fluorescence Polarization Immunoassay For The Detection Of Neomycin

Neomycin belongs to Aminoglycosides.It can be used to treat otitis,dermatitis and other infections of livestock and poultry,as well as some diseases like white diarrhea.Furthermore,it also can be used to prevent animal diseases,promote animal growth and development.It has been widely used in animal husbandry because of its lower price and less consumption.However,long-term medication or overdose will not only causes harm to animals,but also to people through the food chain.Its harmful effects include ototoxicity,nephrotoxicity,neuromuscular block phenomenon and so on.Ototoxicity,renal toxicity and neuromuscular blockade are the main harmfulness.There are many existing detection methods,such as microbiological methods,chromatographic methods,immunological detection methods and so on.Microbiological methods can be used to analyze the veterinary drug residues in food by semi-quantitative screening experiment,but it can not be quantitatively determined;thin layer chromatography can separate a variety of substances at low cost,but the results are susceptible to subjective factors;high performance liquid chromatography can provide necessary detection specificity and sensitivity for pharmacokinetic studies and other investigative studies,but requires expensive equipment and skillful operators;rapid enzyme-linked immunosorbent assay is very useful in the accurate determination of neomycin levels in complex systems,but the need for repeated washing and incubation during operation has affected the detection time and efficiency to a certain extent.Therefore,it is a hot research to build a high-throughput detection method that is simple,sensitive,fast and efficient.In this paper,a new method for the detection of neomycin,fluorescence polarization immunoassay has been developed.It is a kind of homogeneous detection methods.The method was developed by synthesising a conjugate called tracer by coupling the fluorescent substance with the small molecule,preparing the artificial antigen,and selecting the monoclonal antibody by cell fusion,finally to detect the neomycin in milk.This article contains the following sections:1.Synthesis and identification of neomycin artificial antigenAnalyze neomycin chemical structure,direct at amino group of neomycin,select bovine serum albumin(BSA)and ovalbumin(OVA)as a carrier protein to prepare artificial immunogen NEO-BSA and NEO-OVA by glutaraldehyde method.The artificial antigens were initial verified by UV-scanning and SDS-PAGE further identified by animal immunization.The results of UV scanning showed that there was no overlap between the coupling product and carrier protein.The results of SDS-PAGE electrophoresis showed that the migration rate of the carrier protein was faster than that of the coupling product,and two experiments were performed to determine the success of artificial antigen coupling.The antiserum titer of NEO-BSA mice was more than 1:6400.The half inhibitory concentration(IC50)is 27.35 ng/mL,showing that the artificial antigen owns good immunogenicity,and can induce the animal body to produce specific antibody of neomycin.2.Preparation and identification of monoclonal antibody against neomycinThe spleen cells with the highest titer and the highest sensitivity in mice that immunized with NEO-BSA fused of SP2/0 myeloma cells through cell fusion technology.Indirect enzyme linked immunosorbent assay(ELISA)was used to screen and clone hybridoma cells,3 hybridoma cell lines secreting monoclonal antibodies against neomycin were screened,named 1E9?4E8?1G1?The titer of the supernatant was determined to be above 1:6.4×103,and the 1E9 hybridoma cell line with high titer was selected to prepare ascites.Ascites were obtained for required monoclonal antibody purification.The monoclonal antibody was identified by polyacrylamide gel electrophoresis.The linear regression equation was y=-60.946x+59.915 with the correlation coefficient R2=0.9948,according to the linear regression equation,IC50=1.338ng/m L.The specificity was also tested by indirect competitive ELISA,and no cross reaction was found with other drugs.The monoclonal antibody of neomycin with highly specific and sensitive has been successfully prepared,providing the required antibody for the fluorescence polarization immunoassay.3.Establishment of fluorescence polarization immunoassay for the detection of neomycinNeomycin was conjugated with fluorescein isothiocyanate fluorescein(FITC)to obtain tracer,which was separated and purified by TLC.After extraction with methanol and CBS,the best separation was obtained,i.e.Rf=0.4 extracted from carbonate buffer(CBS).The linear regression equation Y=128.9+53.2/[(1+56.5/x)-1.175]was obtained by optimizing the working concentration of the tracer and the antibody,and the calibration curve was established.The IC50 was 56.5ng/mL,the linear rangewas 17.37ng/mL-183.92ng/m L,and the correlation coefficient R2=0.9930.4.Application of fluorescence polarization detection method in detection of neomycin residues in milkOptimize the treatment of milk to reduce the matrix effect: after diluting the milk,use saturated ammonium sulfate to precipitate the protein.Add neomycin to the milk,to prepare standard solution that concentration is 30ng/mL,50ng/m L,70ng/mL.After treating the standard milk,establish the fluorescence polarization immunoassay to detect the Neomycin in milk.The detection rate was 87.91%-114.28%.