【作者】 郭杰标；

【导师】 许杨；

【作者基本信息】 南昌大学 ， 食品科学， 2010， 博士

【摘要】 第一部分基于通用抗体检测伐地那非及其类似物免疫学方法的研究磷酸二酯酶-5（Phosphodiesterase-5, PDE-5）是特异性水解环磷酸鸟苷的酶。通过抑制剂PDE-5,能够提高阴茎海绵体中环磷酸鸟苷的含量,导致阴茎动脉及阴茎海绵体的平滑肌舒张从而获得治疗阳痿的效果。盐酸伐地那非（Levitra,拜尔）是一种获US-FDA批准用于治疗阳痿的PDE-5抑制剂药物。近年来,违法添加到保健品和植物药品的伐地那非及其类似物对公众的健康构成了威胁。不当使用伐地那非会导致头痛、面部潮红、消化不良、视力模糊和肌肉酸痛等副作用。如果病人在服用硝酸酯药物的同时服用含伐地那非的保健品将会造成致命的低血压。已经有两种文献报道的伐地那非结构类似物,其结构式中的发色团结构是伐地那非及其类似物的共有结构。伐地那非类似物具有更强的代谢稳定性和脂溶性,能够在体内长时间维持高血药浓度,并更容易通过血脑屏障产生中枢神经伤害。因此,必须加强对伐地那非结构类似物的监管和筛查。目前,检测伐地那非的主要有HPLC、LC-MS、GC-MS和LC-ESI-MS/MS等仪器检测方法,虽然灵敏、精确和可靠,但需要昂贵设备且运行费用高,难以在基层工作中大规模开展。本研究通过制备以伐地那非及其类似物共同基团为主要抗原位点的人工抗原,获得了能够同时检测伐地那非及其类似物的通用抗体,并建立了同时检测伐地那非及其类似物的免疫学方法。结果如下：1.使用戊二醛利用亲电取代反应原理,合成突出伐地那非及其类似物共同基团的人工抗原；用免疫抗原免疫动物,并利用杂交瘤技术筛选得到能够识别伐地那非及其类似物的通用单克隆抗体（Generic Monoclonal Antibody）;2.建立了同时检测伐地那非及其类似物的免疫学检测方法。对伐地那非的检测限为5.0ng/mL, IC50值为18.2ng/mL;3.该系统检测32个样品种伐地那非及其类似物的结果,与HPLC的检测结果一致无明显差异（P<0.05）。第二部分基于通用抗体检测（氟）喹诺酮总量免疫学方法的研究（氟）喹诺酮是一类广谱抗菌素,通过抑制原核生物的DNA解旋酶对革兰氏阴性和阳性菌发挥高效杀伤作用。近年来,（氟）喹诺酮类药物在临床和兽医中的广泛应用,还被用作食品添加剂促进动物的生长。这类药物的滥用会危害公共健康（如产生过敏反应、细菌耐药性）,污染环境和影响工业生产（如导致乳酪和酸奶发酵无法进行）。因此,欧盟食品安全委员会制定了动物源食品中（氟）喹诺酮类药物的残留限量（96／23／EC）,规定欧盟各国必须对对食品中该类药物的残留量进行监管。中国农业部规定（氟）喹诺酮药物在各种动物源性食品中的残留量不得超100μg／kg（农业部部颁标准,NO.278.2003.5.22）.目前,用于该类药物残留检测的方法有HPLC, LC-MS, GC-MS以及CE等。基于仪器方法的多残留检测需要复杂的样品预处理过程,要借助昂贵的检测设备而且运行费用高。而基于免疫学技术的多残留检测方法具有快速、灵敏、特异、价廉的优点,很适合在基层检测部门进行初筛,使值得关注的一个研究方向。本研究选用环丙沙星（CPFX）连接到经糖基化修饰的BSA作为免疫抗原,连接到OVA作为检测抗原,把“（氟）喹诺酮药物共同基团突出在人工抗原的表面。使用糖基化载体能够提高免疫抗原的免疫效果,用杂交瘤技术筛选对（氟）喹诺酮药物共同基团的通用单克隆抗体,用于开发检测（氟）喹诺酮该类抗菌素的多残留免疫检测方法。结果如下：1、采用戊二醛作为连接臂,将代表药物环丙沙星连接到不同的载体蛋白制备突出（氟）喹诺酮类药物共同基团的免疫抗原和检测抗原。经研究发现采用糖基化BSA作为载体蛋白制备免疫抗原,能够提高免疫原性并获得更好的动物免疫效果；2、应用杂交瘤技术,筛选并鉴别到一株能识别多种（氟）喹诺酮类药物的通用单克隆抗体,以环丙沙星为代表药物绘制竞争抑制曲线,50%竞争抑制的CPFX浓度(IC₅₀)为10.2 ng／mL,最低检测限度为1.0 ng／mL,线性范围在1.0-27ng／mL。对其他10种常用的（氟）喹诺酮类药物的交叉反应率在7.2-96.2%；3、分别在20个鸡肝和20个鸡蛋中添加50、100、150 ng/g的环丙沙星,采用本研究开发的样品处理和ELISA方法检测,加标回收率在64.0-91.8%之间,批内差异在8.7-12.5%。4、采用本研究开发的样品处理和ELISA方法,对20个鸡肝和20个鸡蛋样品进行检测,得到11个阳性结果和29个阴性结果,与HPLC检测结果一致。更多还原

【Abstract】 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.更多还原