Study On Immunoassay Method Of Detecting The Residues Of Fluoroquinolones

Fluoroquinolones (FQNS or FQS) is the third generation quinolones antibiotics, it has high antibacterial activity to Gram-positive bacteria, Gram-Negative bacillus,coccobacteria and gonococci; it also has powerful inhibiting effect to some of the chlamydia, sprirochete, mycoplasma and anaerobic bacterium, it almost applies to all the regular bacteria-infected diseases. On the other hand, because of the widespread application of FQNS, often accompanied with drug-abuse and irrationality of applying, the adverse reactions (ADR) of FQNS is gaining more and more attention. The ADR of FQNS mainly appears in human beings, and it is not serious in animals. However, the residues and the drug-resistant strains, especially intestines strains, in medicated animal will disseminate to human race via food chain. That also has bad effect on people's health. Moreover, FQNS is given to people and animals simultaneously; this will expand the harm caused by drug-resistant strains, will largely shorten the market cycle of the newly developed FQNS. Some of the countries, regions and organization have made stipulation to the using of FQNS and the Maximum Residue Limit(MRL), so it is imperative under the situation to build effective, fast, high-sensitivity methods of detecting the residues of FQNS.Our research, based upon the chemical structure of FQNS, experiments the Norfloxacin(NFLX), Ciprofloxacin(CPLX), Danofloxacin(DFLX) and Sarafloxacin (SALX), which have similar structure with it. We try to find a kind of medicine that can detect the residues of various FQNS and find out the detecting method by making use of the immunology principle and testing the medicine specificity. FQNS is a kind of small molecule material, belonging to a hapten, and only has reactionogenicity, no immunogenicity. We can perform immunological experiment only under the situation that the preparation of complete antigen is reached by coupling it with biomacromolecule.Firstly, using the N-hydroxysuccinimideeater method(NHS), we prepare immunizing antigen by coupling the four medicines respectively with bovine serum albumin(BSA), coating antigen by coupling with ovalbumin (OVA). Thus, we get eight kinds of complete antigen. When we scan the complete antigens, the four medicines, BSA and OVA with ultraviolet light, whose wave-length is between 200～400nm, we can find that the maximum absorption peak of BSA is at 278nm, the maximum absorption peak of OVA is at 277nm, the maximum absorption peaks of NFLX,CPLX,DFLX and EFLX are at 271nm,270nm,275nm and 272nm respectively, the maximum absorption peaks of NFLX-BSA, NFLX-OVA, CPLX-BSA, CPLX- OVA, DFLX-BSA, DFLX-OVA, SALX-BSA and SALX-OVA are respectively at 276nm,274nm,275nm,274nm,277nm,276nm,277nm and 276nm. This indicates that the absorption spectrum of the conjugate are the integration of the absorption spectrum of medicine and protein. The result shows that the medicine has coupled with carrier protein successfully. At the same time, we prepare antiserum by immunizing mice with immunizing antigen, then perform ELISA test on antiserum by coating antigen coating ELISA plate. The results shows that all the titer of the four antiserum reach above 1:32000, this indicates that the antiserum we have got is specificity aiming at medicine, but not carrier protein; we can also prove the coupling is successful when identify the antiserum in immunology.Proceed the ci-ELISA experiments on the four antiserum we have got, and the standard curve and linear regression equations of them are respectively NFLX:y = -18.418x + 94.986(R2 = 0.9843), the limit of detection(LOD) =34.5ng/mL; CPLX:y = -19.386x + 95.648(R2 = 0.9875), LOD=31.3ng/mL; DFLX:y = -18.666x + 94.042(R2 =0.9896), LOD=29.24ng/mL;SALX:y = -18.925x + 91.239(R2 =0.9912), LOD=19.86ng/mL. We have tested specificity (cross reaction rate) of antiserum with the same kind medicine and find that the serological specificity of NFLX antiserum is stronger, the cross reaction rate is lower, the cross reaction of the other three antiserum appears more; of them, the antiserum of SALX has the most cross reaction, especially with NFLX, CPLX and Enrofloxacin (EFLX). Thus, select SALX for further study.Adopt the same method as mentioned above to re-couple SALX and BSA, then prepare immunizing antigen--SALX-BSA, prepare antiserum of SALX by immunizing rabbits. Those will be used for the establishment of the method of detecting the residue of FQNS after we have purified them with ammonium sulfate. The ci-ELISA detecting method was established according to the competitive principle and the detecting condition is optimized. The optimized reaction condition is: The SALX-OVA was diluted to ELISA plate at a concentration of 0.25μg/mL(100μL/well) and incubated 1h at 37℃. Plate were washed three times with PBST for 3 minutes per times(the mode of washings is identical next), and 200μl 5% goat blood serum was added to each well to eliminate nonspecific binding by blocking the plastic surface where protein was not bound. After 2h of incubation at 37℃. washing, and 1:20000 antiserum of SALX(50μL/well) and varying concentrations of standard SALX (50μL/well) were added. After 1.5h of incubation at 37℃. washing, and goat anti-rabbit IgG-HRP (1:3000, 100μL/well) was added and incubated for 1h at 37℃. washing, and OPD substrata solution(100μL/well) was added, followed by the addition of stopping solution(2mol/L H2SO4, 50μL/well) after 15 minutes in the dark at room temperature.Absorbance at 490nm was determined by an enzyme immunoassay reader. Dilute the standard SALX as 40000,4000,400,40,4,0.4ng/mL, then perform the ci-ELISA detection under the optimized condition. Percent inhibition ratio(%B/B0) was calculated from the absorbance obtained in the presence(B) and absence(B0) of SALX in standard. A linear dose-response standard curve was prepared by plotting log [SALX] versus percent inhibition ratio. The linear regression equation of standard curve was y = -17.098x + 88.66(R2=0.996), that accords with the judging standard of linear relation. According to the testing of specificity, the cross reaction rate of the reaction with SALX,NFLX,CPLX and EFLX is above 80%, the non-fluoroquinolones were tested and there was no cross-reaction between them . This method can proceed qualitative detection on SALX, NFLX, CPLX and EFLX, and LOD of them are 19.319ng/m, 22.646ng/mL, 26.181ng/mL and 19.054ng/mL, respectively.Append standard SALX, NFLX, CPLX and EFLX to rabbit musculature to proceed detection. The average recovery rate is above 85% within the range of 0.5~2000ng/mL.The establishment of this method will not only settle the base for the development of ELISA kit, which is used for the detection of FQNS residues, but also supply the effective detecting measure for the supervising departments who are in charge of the medicine residues in animal food. At the same time, it has also explored the ways for the study of the methods of detecting some kind of medicine residues.