Studies On The Preparative Technique Of Enrofloxacin Antiserum And Its Immunoassay Method

Enrofloxacin(EF) is a synthetic antibacterial agent that belongs to the fluoroquinolone group (FQs). It has been widely used in veterinary and fish. With the increasing uses, the FQs residues in animal edible tissues could cause serious public health problems and the detection of it has become more and more important.Now in the world, the common way to determine FQs residues is chromatography, but it is both labor-intensive and expensive. So a simple and reliable analytical method for this drug is required. Immunoassay screening methods have been recently developed as an alternative to the traditional methods.The purpose of the study here is to synthesize and to identify the EF complete antigens, which are then immunized to BALB/C rats. On the basis of the polyclonal antibodies ( PcAbs ) , an indirect competitive enzyme-linked immunosorbent assays (Ci-ELISA) is preliminary developed to monitor the residues of EF in animal edible tissues. The experiment includes three parts: I The synthesis and identification of complete antigens, including the treatment of carrier protein by ethyl diamine (EDA), the contrast between different coupled methods to different proteins, the optimization of the conjugation by orthogonal test and the identification of complete antigens by UV spectrum, ratio, the character of antiserum and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS). Ⅱ The production and determination of antiserum. BALB/C rats are immunized with different antigens to produce antiserum and an ELISA is used to determine the titers and the specificities. ⅢAn Ci-ELISA method is preliminary established and evaluated the precision, accuracy and sensitivity. All ELISA results areconfirmed by using HPLC which is used as the routine assay. The results are as followed:1. It is the first time to treat the carrier proteins by using EDA and it is found that coupling effects could be obviously improved.2. The results of the orthogonal test showed that the affect of the carrier protein is the most appreciable in the three factors and when ovalbumin( OVA) is used as carrier protein, the mole ratio is EF: protein =75:1 and the conjugation time is 24h, the titer of serum can be the highest.3. To various carrier proteins, the coupling methods are also different, active ester method is better to OVA while carbodiimide method is better to bovine serum albumin (BSA) .4. The antiserum titer obtained by using ELISA is higher than 250 000 when the carrier protein is BSA, while the titer can reach 300 000 when OVA is used as carrier protein. In Ci-ELISA assay, four quinolone derivatives show high cross-reactivity with EF about 14.2% ~ 161.7%(BSA is used as protein) and 1.3%—169.8%(OVA is the protein), but for the other four antibiotics the cross-reactivity is both lower than 0.01%.5. Precision of the Ci-ELISA employed to detect EF indicated by coefficients of variation of intra-assay and inter-assay are 9.8%~17.4% andll.9%~23.4% respectively, the mean recoveries of EF is 60.07%-— 120.8%.The limit of detection(LOD) is lng/mL and the linear range is lng/mL— lOOOng/mL.6. The HPLC and ELISA results show close correlation.Now the research on conjugation of complete antigen is limited and there is few information about the optimization of FQs-protein conjugates. Our research will provide basis on the development of rapid detection of FQs in animal edible tissues.