【作者】 王瑞；

【导师】 黄登宇；

【作者基本信息】 山西大学 ， 食品科学， 2015， 硕士

【摘要】 硝基呋喃类药物及其代谢物具有强毒性和致癌致畸性,残留于动物源性食品中会对人体健康产生巨大危害。因此,许多国家已将硝基呋喃类药物列为禁用药物,并规定在动物源性食品中硝基呋喃类药物及其代谢物不得检出。但由于其成本低廉,药效明显,仍有不法分子为牟取暴利继续使用。呋喃妥因是硝基呋喃类药物之一,目前,针对呋喃妥因代谢物(AHD)残留的检测多采用仪器检测分析法,这些方法虽然准确、灵敏,但需要昂贵的设备和专业技术人员操作,而且耗时长,成本高,不适用于大量样品的快速筛查。因此,建立一种便捷、快速、灵敏的AHD残留检测方法,对于迅速控制食品安全风险,保障人民群众饮食健康安全具有重要意义。本实验建立了检测AHD残留的酶免疫分析法,即化学发光酶免疫分析法(CLEIA)和酶联免疫分析法(ELISA)。首先,采用对醛基苯甲酸将AHD衍生化为半抗原CPAHD,并在此基础上采用活化酯法将CPAHD与卵清蛋白(OVA)偶联合成包被原；分别用活化酯法和混合酸酐法将CPAHD与辣根过氧化物酶(HRP)偶联合成了两种酶标抗原。通过直接竞争ELISA法,优选出采用活化酯法制备的酶标抗原灵敏度更高,更加符合CLEIA法和ELISA法的实验要求。其次,建立了AHD的间接竞争CLEIA法和直接竞争CLEIA法,并对化学发光液、包被条件、封闭液和竞争时间等参数进行了优化,间接法中优选了包被原与抗体的最适稀释度、直接法中优选了抗体与酶标抗原的最适稀释度。结果表明,间接竞争CLEIA法和直接竞争CLEIA法的IC50分别为0.753ng/mL和0.539ng/mL;特异性良好,除对呋喃妥因原药存在一定交叉反应外,对其他结构类似物及衍生化试剂均无交叉反应；准确性和精密度良好,对猪肉、鸡肉和鱼肉样品的添加回收率在84.9%-103.4%之间,批内和批间变异系数均小于12%；间接竞争CLEIA法对猪肉、鸡肉和鱼肉的最低检测限分别为0.020、0.021和0.019μg/kg,直接竞争CLEIA法对猪肉、鸡肉和鱼肉的最低检测限分别为0.013、0.013和0.012μg/kg。第三,建立了AHD的间接竞争ELISA法和直接竞争ELISA法,并对显色时间进行优化,其余优化条件项目与CLEIA法相同。结果表明,间接竞争ELISA法和直接竞争ELISA法的IC50分别为2.186ng/mL和1.291ng/mL;特异性良好,除对呋喃妥因原药存在一定交叉反应外,对其他结构类似物及衍生化试剂均无交叉反应；准确性和精密度良好,对猪肉、鸡肉和鱼肉样品的添加回收率在82.6%～104.7%之间,批内和批间变异系数均小于13%；间接竞争CLEIA法对猪肉、鸡肉和鱼肉的最低检测限分别为0.219、0.214和0.208μg/kg,直接竞争CLEIA法对猪肉、鸡肉和鱼肉的最低检测限分别为0.115、0.113和0.109μg/kg.实验结果表明,本实验建立的AHD酶免疫分析法,其灵敏度、准确性、精密度及最低检测限均能满足实际样品检测的需求。其中CLEIA法的灵敏度和最低检测限均优于ELISA法,直接竞争CLEIA法又比间接竞争CLEIA法具有更高的灵敏度和更低的检测限,可为一线食品安全监管人员检测AHD残留量提供一种准确、可靠、便捷的方法。结合前期建立的对食品小样本的灰评估方法,可以从点和面等不同维度较为全面地判别食品风险程度,对有效和积极防控食品安全重大隐患具有较强的实际应用价值。更多还原

【Abstract】 Nitrofurans and their metabolites are a class of compounds with strong toxicity, carainogenicity and teratogenicity. It will have a great harm to human health when those compounds remained in anaimal-derived food. As a consequence, many countries have classified nitrofurans as banned drugs. But due to its low cost and efficacy significantly, there are still lawless elements to reap huge profits to continue to use them. Nitrofurantoin is one of nitrofurans. At present, various instrumental analysis methods with high analytical precision have been used to detect the residue of AHD. However, these methods require expensive equipments and professional and technical personnel to operate and also time-consuming and costly. Thus, the establishment of a convenient, rapid and sensitive method for detecting residues of AHD in anaimal-derived food will have a great significance to the effective control of food safety risk and ensure the safety of people’s diet. In this study, CLEIA and ELISA methods for AHD were developed.The hapten CPAHD was synthesized by conjugating AHD with 4-CBA. Then made it conjugate with OVA by using N-hyduoxysuccinimide ester method to synthesize coating antigen. The enzyme-labeled antigen was synthesized by N-hyduoxysuccinimide ester method and mixed anhydrides method respectively. Then this two kinds of enzyme-labeled antigen were identified by dc-ELISA. The results indicated that the one synthesized by N-hyduoxysuccinimide ester method has a higher sensitivity.The indirect competitive CLEIA and direct competitive CLEIA for AHD were established. Then series of test parameters, such as chemiluminescent solution, coating condition, blocking liquid and competitive reaction time were optimized. Besides, coating antigen concentration and antibody concentration were optimized in indirect competitive CLEIA. Antibody concentration and enzyme-labeled antigen concentration were optimized in direct competitive CLEIA. The results indicated that the IC50 of indirect competitive GLEIA and direct competitive CLEIA were 0.753ng/mL and 0.539ng/mL. The cross reactivity studies showed that the CLEIA had good specificity for AHD and there was negligible cross reactivity with other structurally related compounds except nitrofurantoin. Recoveries rates of AHD ranged from 84.9% to 103.4% in fortified pork, chicken and fish samples. The intra and inter assay coefficient of variation were less than 12%. The detection limit of indirect competitive CLEIA for pork, chicken and fish samples were 0.020,0.021 and 0.019μg/kg respectively. While the detection limit of direct competitive CLEIA for pork, chicken and fish samples were 0.013,0.013 and 0.012μg/kg respectively.The indirect competitive ELISA and direct competitive ELISA for AHD were also established in this study. The developing time was optimized. And the rest of optimization conditions were set the same as the method of CLEIA. The results indicated that the IC50 of indirect competitive ELISA and direct competitive ELISA were 2.186ng/mL and 1.291ng/mL. The ELISA had good specificity for AHD and there was negligible cross reactivity with other structurally related compounds except nitrofurantoin. Recoveries rates of AHD ranged from 82.6% to 104.7% in fortified pork, chicken and fish samples.The intra and inter assay coefficient of variation were less than 13%. The detection limit of indirect competitive ELISA for pork, chicken and fish samples were 0.219,0.214 and 0.208μg/kg respectively. While the detection limit of direct competitive ELISA for pork, chicken and fish samples were 0.115,0.113 and 0.109μg/kg respectively.These results indicated that the CLEIA and ELISA methods with good sensitivity, precision, accuracy and detection limit can meet the need of detecting AHD residues in actual samples. The sensitivity and detection limit of CLEIA methods were better than ELISA methods. Meanwhile, compared with indirect competitive CLEIA method, direct competitive CLEIA has higher sensitivity and lower detection limit. It can provide an accurate, reliable and convenient way for grassroots food safety regulators to detect AHD residues. Combined with the gray evaluation method for a small sample of food, which was established early, it can be more fully to determine the risk extent of food from different dimensions. This initiative has a strong practical value for effective and positive prevention and control of major food safety risks.更多还原