Development Of Immunoassays For Vardenafil (and Its Analogues) And For The Total (Fluoro) Quinolones Based On Generic Antibodies

Part 1:Research on an immunoassay for simultaneous detection of vardenafil and its analogues based on generic antibodyPhosphodiesterase-5 (PDE-5) inhibitors can inhibit the PDE-5 enzyme thus raising the cyclic guanosine monophosphate (cGMP) levels causing a vasodilating effect.Vardenafil is one of the licensed PDE-5 inhibitors for the treatment of erectile dysfunction. Undeclared vardenafil and related analogues adulterated in herbal products are a threat to public health. First, vardenafil has a series of adverse effects such as headache, facial flushing, dyspepsia, visual disturbances and muscle aches. Secondly, the interaction between PDE-5 inhibitors and certain drugs containing nitrates may cause drastically lower blood pressure. Up to date, there were two analogues of vardenafil that have been elucidated in dietary supplements. The chromopore group is the common structure of vardenafil and its analogues. The analogue of vardenafil has a better metabolic stablity and fat solubility, so it can be meantained in a higher concentration in vivo for a long time and is prone to pass through the blood brain barrier to cause injury to the central nervous system. Therefore, there is an urgent need to develop reliable analytical methods to screen illegal additives of vardenafil and its analogues in herbal commodities.HPLC,LC/MS,GC/MS and LC-ESI-MS/MS are the methods in common use for the analysis of vardenafil and its analogues in herbal products fluids. The above instrumental analyses, although accurate and reliable, have disadvantages of being time-consuming and costly, relying upon expensive instruments and sophisticated operators. In this study, attempts have been made to generate a monoclonal antibody (McAb) that can recognize the chromophore structure of vardenafil. By using a McAb, an indirect competitive ELISA (ic-ELISA) could be established for the detection of not only vardenafil but also its potential analogues as illegal additives on herbal products in a simplified and sensitive way. The results are decribed as follow:1. Vardenafil was linked to the carrier proteins by using glutaraldehyde as a bridging reagent in pH5.0, leaving the chromophore structure of vardenafil exposed as a major antigenic site. The Balb/c mouse immunized by vardenafil-cBSA was selected for fusion. Eight hybridomas were screened and identified as being specific to vardenafil. McAb of 4B9 was characterized as being specific to the common structure of vardenafil and its analogues.2. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established based on this McAb, the limit of detection of vardenafil was 5.0 ng/mL, with an IC50 value of 18.2 ng/mL.3. The ELISA method was in good agreement with LC-UV when detected 32 herbal products containing vardenafil and its analogue piperidenafil (P<0.05).Part 2:Research on an immunoassay for the detection of total (Fluoro)quinolones based on generic antibody(Fluoro)quinolones are a class of effective antibiotics against Gram(+) and Gram(-) bacteria via the inhibition of the DNA gyrase, they are widespread applied as both human and veterinary medicine, as well as feed additives at sub-therapeutic levels to promote the growth of animals. The misuse of this class of drugs may give rise to public health (e.g. allergic reactions, antibiotic resistance, etc.), environmental and industrial (e.g. cheese or yoghurt production, etc.) problems. Therefore, in agreement with the Council Directive 96/23/EC, the European Union (EU) countries must monitor the presence of these drugs residues in live animals and animal products. In China, total maximum residue limit (MRLs) of FQs in foodstuffs was set of 100μg/kg (Ministry of Agricutrue of the P.R. China, NO. 278.2003.5.22).Nowaday, instrumental analyses, including HPLC, LC-MS, GC-MS and CE, are the main methods for the determination of generic FQs in food and environmental samples. These methods, mainly relying on complicated clean-up procedures and expensive instruments, had disadvantages of being time-consuming and costly. Recently, multi-residue immunoassays have been the centre of interest for determination of a class of related small molecular analytes.In this study, Ciprofloxacin (CIP) were applied as the representative drug of (Fluoro)-quinlones for generation of antibodies against this class of compounds. The bifunctional reagent of Glutaraldehyde was employed as a bridging reagent to link the secondary amine group of CIP to different carrier proteins for the preparation of immunogens and coating conjugates, respectively, leaving the group of'4-quinolone carboxylic acid'exposed in both conjugates as a major antigenic site. A better immunological effect for generation of anti-CIP antibodies was observed by using a glycosylated protein as carrier of immunogen.By using this synthesis strategy, a McAb with broad specificity against quinlones and FQs, and established a multi-residue indirect competitive ELISA for the analysis of this class of analytes. The results were described as follow:1. Glutaraldehyde was employed as a bridging reagent to link the secondary amine group of CIP to different carrier proteins for the preparation of immunogens and coating conjugates, respectively, leaving the group of'4-quinolone carboxylic acid'exposed in both conjugates as a major antigenic site. A better immunological effect for generation of anti-CIP antibodies was observed by using a glycosylated protein as carrier of immunogen.2. By using hybridomas technique, a monoclonal antibody with ability of reconizing several (fluoro)quinolone drugs was obtained. The standard curve was prepared with ciprofloxacin, the IC50 value was 10.2ng/mL and the limit of detection (LOD) was 1.Ong/mL. To the other 10 common used (fluoro)quinolone drugs, the cross-reactivities were ranging from 7.2%to 96.2%.3. Twenty chicken liver and 20 chicken egg samples, which were proved not containing (fluoro)quinolones, were fortified with ciprofloxacin in three levels of 50,100 and 150ppb. By using the sample preparating method and ELISA system developped in the current study, the recoveries of detecting the fortified samples were ranging from 64.0%to 91.8%, with a range of coeffecient of variablity being 8.7-12.5%.4. Twenty chicken liver and 20 chicken egg samples collected from local market were determined by the current ELISA system and HPLC method, respectively. Elevant samples were detected having residue of (fluoro)quinolones. And the 11 positive samples were confirmed containing different (fluoro)quinolones, while the 29 negative samples were confirmed not containing (fluoro)quinolones, by using HPLC determination.