The Study And Establishment Of Enzyme-linked Immunoassay Method Of Quinolone Residues

Quinolones are an important class of group synthetic antibiotics. The mechanism of quinolones involves theirs acting on the bacterial nucleus directly, inhibiting the activity of DNA–gyrase, and then abolishing its activity by interfering with the DNA rejoining to destroy the metabolism and multiplication of bacteria, finally inducing the death of bacteria. Originally they were used for the urinary tract infection, widely applied to prevent and treat of various diseases in animal husbandry and aquaculture.The NOR derivative was synthesized by alkylation characterized by UV, LC–MS and H1NMR, and the production was coupled to bovine serum albumin (BSA) and ovalbumin (OVA) to synthesize immunogen NOR derivative–BSA and coating antigen NOR derivative–OVA respectively by glutaraldehyde method. Besides five QNs were directly coupled to BSA and OVA by active ester method. Conjugates were identified by the method of ultraviolet and visible light scanning. Then polyclonal antibodies were acquired from animal immunization of the antigen. After screening, the antiserum reached dilute rate 1:102400 was selected, through a indirect enzyme–linked immunosorbent assay.Based on this antiserum a polyclonal antibody–based enzyme–linked immunosorbent assay has been developed, optimized and validated to measure QNs. Influence of several physicochemical parameters, such as coating buffer, antibody dilution buffer, blocking buffer etc. were selected to provide a highest sensitivity on the ELISA format.After optimization, the standard curve was obtained and the detection limit of the assay is as low as 0.0126ng/mL. Cross–reactivity studies demonstrated that the quinolones possessed similar structurally relations are recognized to different extent (2%–112%) which proves the high group–specificity of the assay. The recoveries were between 63% and 95%, with the coefficients of variation lower than 20%. The validation of the method developed showed a good reliability and accuracy. Therefore, this indirect ELISA method is suitable for the rapid screening of the quinolone multiresidues in animal food.Simultaneously, a HPLC–ESI–MS/MS method was also developed for verification of 19 QNs in animal muscle matrices. By detecting the recoveries and coefficient variation of quinolones in fortified samples, it shows that this method completely accords with the detection requirements of the veterinary drug residue in animal food at home and abroad. Keyword: quinolones; polyclonal antibody; ELISA; multiresidue...