Study On Rapid Immunoassay For Detection Of Flumequine In Animal Foods

Flumequine(FLU)is an animal-specific quinolone antibiotic used to prevent and treat diseases in livestock,poultry and aquatic animals.However,the abuse or over standard use of FLU often occurs,resulting in the residual problem in animal foods,which causes great harm to human health.Every country in the world has stipulated the maximum residues of FLU in animal foods as the standard and basis of food safety supervision.It is also necessary to establish simple,rapid and high-throughput methods for detection.In this study,flumequine complete antigen was synthesized by active ester method.Polyclonal antibody was prepared by immunizing animals.An indirect competitive enzyme-linked immunoassay(icELISA)for flumequine detection was established.Solid phase membrane-based immunoassay for flumequine detection was established.By using prepared flumequine molecular imprinting membrane as artificial antibody,a direct competitive biomimetic enzyme-linked immunoassay(BELISA)was established.The established icELISA and BELISA methods were suitable for rapid quantitative screening of a large number of samples with simple pretreatment,fast operation and low detection limit.Solid phase membrane-baced immunoassay was suitable for rapid qualitative screening,accurate,reliable and easy to operate.Synthesis of complete antigen of flumequine and preparation of polyclonal antibody.FLU-BSA(immunogen)and FLU-OVA(coating antigen)were prepared by coupling FLU hapten with carrier protein bovine serum albumin(BSA)and ovalbumin(OVA)by active ester method.The coupling results were tested by ultraviolet spectrum scanning and SDS-PAGE gel electrophoresis and showed that the artificial antigens were synthesized successfully.Two New Zealand white rabbits were immunized with immunogen FLU-BSA to obtain two antiserum(FLU-1 and FLU-2).The titer and affinity of FLU-1 and FLU-2 were determined by indirect competitive ELISA.The titer of FLU-1 was the same as that of FLU-2(1:12800).After purification,the affinity of FLU-1 antibody(IC50 was 0.06 ng/mL)was significantly higher than that of FLU-2(IC50 was 0.21 ng/mL).Therefore,FLU-1 antibody was selected for subsequent immunoassay.Establishment of icELISA method for determination of flumequine residues.Indirect competitive ELISA was used to optimize the detection conditions of FLU.The optimum conditions were as follows:coating amount was 10 ?g/mL,antiserum dilution was 1:3200,1%gelatin was used as blocking solution,dilution of enzyme-labeled second antibody was 1:2500,pH was 7.5,reaction buffer was 1×PBS,acetonitrile content was 0%?20%.Under the optimum conditions,an indirect competitive ELISA method was established.The IC50 of the method was 2.03 ng/mL and the detection limit was 1.21×10-4 ng/mL.The method had high accuracy and sensitivity.The cross-reactions between FLU and its structural analogues levofloxacin(LVX),gatifloxacin(GAT)and ofloxacin(OFX)were tested.The cross-reactions were very low and the antibody specificity was good.Pork,beef,shrimp,milk,raw pig liver and cooked pig liver were selected to investigate the elimination method of matrix effect.After ultrasonic extraction,pork,beef and milk were diluted 10 times,shrimp was diluted 20 times and pork livers were diluted 40 times by PBS to eliminate the matrix effect effectively.In the recovery test of standard addition,the recovery rates of the three standard addition levels were ranged from 72.80%to 97.30%.For the validation of the established indirect competitive ELISA,samples were analyzed by HPLC.The results of the two methods were highly fitted,and the correlation coefficient R2 was greater than 0.96.It was showed that the established ELISA method was accurate,reliable,simple and rapid,and could be used for rapid quantitative analysis of flumequine residues in animal foods.Establishment of solid phase-based membrane immunoassay for determination of flumequine residues.Colloidal gold was prepared by trisodium citrate reduction method and immune gold was prepared by labelling FLU antibody with colloidal gold.Nitro-cellulose(NC)membrane strip which used as solid carrier was separately coated with FLU-OVA(as test line)and enzyme-labeled secondary antibody(as control line).The optimum conditions were as follows:blocking solution was 5%skimmed milk,enzyme-labeled antibody dilution times was 40 times,coating amount was 1.0 g,immunogold dilution was 1:5(v/v),the mixing ratio of gold-labeled antibody and the solution to be tested was 1:5(v/v).Under the optimum conditions,the FLU colloidal gold labeled immunochromatographic solid phase membrane was prepared with a minimum detection limit of 40 ?g/L.Matrix effect elimination tests were carried out on samples of pork,beef,shrimp,milk,raw pig liver and cooked pig liver.The matrix extract of pork,beef and shrimp needed to be diluted 20 times,milk and pig livers needed diluted 40 times,with 1xPBS buffer,the matrix effect could be eliminated.In the standard addition recovery test,the obvious gradient could be observed by naked eye,which showed that the method was effective.The established solid phase membrane-based immunoassay was a kind of qualitative detection method.In the actual detection work,there was no need for instruments,and the visual results could be obtained in a short time.It was suitable for rapid qualitative screening of large quantities of samples on the spot.Establishment of BELISA method for determination of flumequine residues.The optimum molar ratio of flumequine to methacrylic acid(MAA)was determined to be 1:2 by molecular dynamics simulation.Molecularly imprinted polymer films(MIPs)were prepared by using FLU as template,MAA as functional monomer,ethylene glycol dimethacrylate(EGDMA)as cross-linker,azodiisobutyronitrile(AIBN)as initiator and 96-well plate as solid phase carrier.It was characterized by static adsorption test and adsorption kinetic analysis and the results showed that the molecularly imprinted polymer films had been successfully synthesized.Enzyme-labeled antigen was prepared by active ester method.The optimum conditions of BELISA reaction system were determined as follows:dilution times of enzyme-labeled antigen was 8000 times,pH was 7.1,and buffer was PBST with methanol content of 5%.Under the optimum conditions,a direct competitive BELISA method was established with the detection linear range of 1?100000 ng/mL,the sensitivity IC50 of 140.62 ng/mL and the detection limit of 1.09 ng/mL.Cross reaction rate of levofloxacin(LVX),ofloxacin(OFX)and enoxacin(ENX)was 17.8%,17.5%and 11.0%respectively,which indicated that the method had high specificity.Matrix elimination was carried out by direct dilution method and the results showed that shrimp sample extract was diluted 20 times and beef sample extract diluted 40 times.Sample recovery rates at three levels ranged from 80.7%to 92.3%,which indicated that the sample pretreatment method was feasible.The accuracy of the method was validated by HPLC.The results showed that there was a good linear relationship between HPLC and BELISA.The correlation coefficient R2 was above 0.98,which indicated that the established BELISA was accurate and reliable,and it could be used for the rapid detection of FLU residues in animal foods.