球虫病是由原虫中艾美耳球虫引起的家禽肠道寄生虫病，在全世界家禽养殖业中广泛存在，导致畜禽生长速度减慢、饲料转化率降低、肉品质下降，每年给家禽业造成数百万美元的经济损失[1]。艾美耳球虫有一个口腔-粪便生命周期，没有中间宿主，根据暴露程度和环境因素，可以引起宿主广泛的发病和死亡[2]。Levine[3]首次发现磺胺类药物可以治疗鸡球虫病, Grumbles等[4]用磺胺喹
抗球虫药物常作为饲料添加剂使用，饲料污染的情况时有发生[6]。这可能会对非目标动物造成毒性作用，并可能导致食物中含有药物残留，在食用抗球虫药物残留的食品后，虽然未观察到对人体产生急性毒性，但长期暴露于低浓度水平的药物残留，人体内可能发生慢性毒性作用[7-8]。因此，许多国家对动物使用该类药物以及在动物源食品中的残留情况进行了严格监控。包括中国(表 1)和欧盟[7]在内多个国家或组织制定了相关法规，规定鸡组织中抗球虫药物的最高残留限量(MRLs)。
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表 1 中国规定可食用动物组织中抗球虫药的MRLs Table 1 Chinese legislation of MRLs for anticoccidial drugs in edible animal tissues |
目前，对抗球虫药物的检测方法主要有色谱(串联质谱)检测法[9-12]，以及免疫分析检测法。免疫检测法是基于抗原-抗体相结合的原理，采用不同的信号放大系统对待测物进行定性和定量。抗体的制备是决定检测技术灵敏度的关键因素。免疫分析方法具有开发简便、成本低、操作简单、能够快速检测的优点，可实现高通量快速筛选，在畜禽养殖场中广泛应用[13-14]。但其无法达到对多种残留药物的同时定量检测。一个成功的免疫检测法必须要具备3个要素：性能优良的抗体、灵敏且专一的标记物和高效的分离手段。传统的单克隆抗体制备过程繁琐，成为免疫分析方法的限制因素之一。
1 酶联免疫吸附分析方法酶联免疫吸附分析(enzyme linked immunosorbent assay，ELISA)是将抗原或抗体结合到固相载体表面，加入待检测的样品溶液以及某种酶标记的抗体或抗原，使其与固相载体上的抗体或抗原反应。再通过洗涤去除抗原-抗体复合物以外的物质。加入酶反应底物显色，结合在固相载体上的酶量与样品中待测物的量成一定比例[15-17]。该方法可用于诊断疾病和检测小分子化合物[18]。其原理如图 1A所示。Tian等[19]描述了一种基于免疫磁珠(IMB)净化的间接竞争酶联免疫吸附法(icELISA)，检测鸡肉和鸡肝中盐霉素的残留。将单克隆抗体固定在羧化微球(直径2.8 μm)的表面上，样品经简单提取后，待测物被抗体特异性吸附，通过磁分离去除上清，保留在磁珠上的待测物随后被洗脱释放。与传统的亲水亲脂平衡柱(HLB柱)净化的icELISA方法相比，该方法的抑制率和灵敏度都有所提高，并且磁珠可重复使用。Li等[20]描述了基于单克隆抗体的icELISA方法，检测可食用鸡组织(肌肉、肾、肝、皮肤和脂肪)中的莫能菌素残留。该方法与高效液相色谱串联质谱法(HPLC-MS)相比，样品检测结果成正相关。这些结果表明，作者制备的单克隆抗体和开发的检测方法是检测鸡组织中马杜霉素的有用工具。Li等[21]制备了乙氧酰胺苯甲酯的单克隆抗体，建立icELISA方法检测鸡肌肉和肝中药物残留，该方法的IC50为0.66 ng·mL-1，对已发表的方法提供了一种节省成本的替代方法。Zhang等[22]使用新型抗体开发一步式icELISA法，可快速、特异性地检测动物源食品中地克珠利残留。Song等[23]建立了基于磁珠净化的icELISA检测方法，检测鸡组织中马杜霉素的残留量，并对磁珠的性能进行了评估。结果表明，免疫磁珠可以快速、稳定地净化样品，并且基于免疫磁珠的ELISA检测方法更简便、省时且环保，可广泛应用于鸡组织中马杜霉素的残留检测。ELISA在检测抗球虫药物残留的应用见表 2。
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A. 酶联免疫吸附分析；B. 免疫层析法；C. 荧光偏振免疫分析；D. 生物传感器 A. Enzyme-linked immunosorbent assay; B. Immunochromatography; C. Fluorescence polarization immunoassay; D. Biosensor 图 1 几种免疫检测分析方法原理 Fig. 1 Principles of several immunoassay methods |
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表 2 ELISA法检测抗球虫药物残留的应用 Table 2 Application of ELISA in detection of residues of anticoccidial drugs |
免疫层析法(immunochromatography, ICA)是一种快速有效的检测方法，该方法大大缩短了整个分析时间, 已应用于多个检测领域，可在非实验室环境下操作[24]。与其他较复杂的分析方法相比，该方法的灵敏度较低[25]。免疫层析分析方法结合了特异性抗体、胶体颗粒(金、乳胶、碳等)和层析膜(硝化纤维素膜)。其原理如图 1B所示，采用免疫标记技术将胶体颗粒直接或间接地与特异性抗体结合，标记抗体涂抹在层析膜上，层析膜一端浸润样品溶液，通过层析作用与抗体特异性结合。测试结果可根据T线(test line)和C线(control line)的显色程度在几分钟内通过目测或仪器测量确定[26]。
最常见的方法是将单克隆抗体与胶体金(colloidal gold, CG)偶联制备免疫层析试纸条，检测样品溶液中药物的残留[27-34]。胶体金具有制备稳定、摩尔消光系数高、颜色明显的特点[35-36]。胶体金免疫层析试纸条制备简便，使用方便，不需配备昂贵的设备和专业的技术人员，因此，能够在畜牧养殖场中广泛应用。Fitzgerald等[37]建立了多残留检测的测流免疫分析方法，通过化学偶联法在特异性抗体上连接有色的羧化微球，可同时检测鸡蛋中常山酮、妥曲珠利和地克珠利3种药物的残留。采用像素分析的方式，通过测量检测区域带的像素强度，将该方法从一步定性转变为可量化分析，简单且经济高效，为将来更有效地同时监测小分子药物铺平了道路。免疫层析法在检测抗球虫药物残留的应用见表 3。
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表 3 免疫层析法检测抗球虫药物残留的应用 Table 3 Application of Immunochromatography in detection of residues of anticoccidial drugs |
荧光偏振免疫分析法(fluorescence polarization immunoassay，FPIA)原理(图 1C)是荧光标记抗原与特异性抗体结合形成大分子复合物后，使荧光素的荧光偏振信号强度增强，当目标待测物存在时，待测物与抗原竞争性结合特异性抗体，使荧光偏振值减小[38-39]。此反应是均相反应，减少了多次洗涤的步骤和反应时间，并且不受溶液颜色和仪器灵敏度变化的影响[40]。该方法常用来检测小分子化合物在样品中的含量[41]，并且可用于现场检测[42]。
Wang等[43]建立并优化了用特异性多克隆抗血清检测鸡组织(脂肪和肌肉)和鸡蛋中马度米星的荧光偏振免疫分析法(FPIA)。采用N-羟基琥珀酰亚胺活性酯法合成荧光标记物来标记马度米星(示踪剂)，并用薄层色谱法(TLC)纯化。该方法的IC50值比最敏感的ELISA方法低4倍。同一团队开发并优化了测定缓冲液中盐霉素的FPIA，表明所用的抗盐霉素单克隆抗体与其他药物无明显交叉反应，与甲基盐霉素的交叉反应率为67.6%，并预测该分析方法可用于食品样品筛选，但还未证实[44]。
时间分辨荧光免疫测定法(time-resolved fluoroimmunoassay, TrFIA)是利用镧系络合物, 如铕(Eu)、铽(Tb)、钐(Sm)和镝(Dy)的三价稀土离子及其螯合物作为标记物的免疫分析技术[45]，镧系络合物很稳定，具有荧光寿命长、stokes位移大、驰豫时间长且能够克服背景吸收的优势[46]。TrFIA旨在降低非特异性背景荧光的干扰，解决放射性同位素使用时的问题, 使设计超灵敏的生物分析方法成为可能性[46-48]，具有安全、费用低、操作简便、可实现床旁快速检测的优势[49-50]。
Peippo等[51]建立了快速TrFIA检测法，用于筛选家禽肌肉和鸡蛋样品中是否存在甲基盐霉素和盐霉素，由于甲基盐霉素与盐霉素具有100%的交叉反应，所以Peippo等仅评估了甲基盐霉素的检测性能。此方法的一个缺点是需要对肌肉样品使用SPE柱进行额外的固相萃取的纯化步骤。以此为基础，Peippo等[52]对前处理方法进行进一步优化，建立了家禽血液和肌肉中甲基盐霉素的TrFIA检测法以及进行血液中的动力学研究。Hagren等[53]将时间分辨荧光测定法与新型的一体化干燥化学概念相结合来进行进一步创新，检测鸡肝和鸡蛋中尼卡巴嗪的残留量。将抗尼卡巴嗪的特异性抗体固定在单个微量滴定孔中，并用绝缘层覆盖。样品提取液自动加入干燥的微量滴定孔中，结果可在18 min内获得。该方法快速、简单，可用作高通量筛选方法。Godoy-Navajas等[54]描述了一种新型的荧光测定技术并运用了新的替代标签检测牛奶中的莫能菌素，示踪剂由功能化纳米颗粒与抗体组成。使用纳米颗粒进行长波长荧光测量，可以提供光谱选择性从而避免样品基质的干扰。实践证明，该方法具有自动化程度高、样品和试剂体积小等优点。
相关方法的总结见表 4。
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表 4 FPIA和TrFIA法在检测抗球虫药物残留中的应用 Table 4 Application of FPIA and TrFIA in detection of residues of anticoccidial drugs |
生物传感器(biosensor)是一种将生物识别元件(酶[55-56]、抗体[57]、抗原、细胞、核酸等)、理化换能器和信号放大装置紧密结合的仪器[58](图 1D)。生物传感器的特点是生物分子之间的特异性结合，例如抗原与抗体，底物与酶，受体与其特异性配体，为了检测这些生物分子之间的相互作用，生物传感器使用感测装置或换能器将生物信号转换成电信号，该电信号由处理器放大、存储和量化[59]。对于兽药残留检测，最常用的生物元素是抗体-抗原亲和对，在这种情况下，生物传感器称为免疫传感器[60]。Huet等[61]详细地描述了用不同类型的生物传感器检测和定量抗微生物化合物。
抗球虫药物的免疫传感器检测方法报道较少。McCarney等[62]用表面等离子体共振(SPR)生物传感器检测鸡肝和鸡蛋中尼卡巴嗪的残留量。他们使用DNC(尼卡巴嗪的组成成分之一)的结构模拟物在兔中产生抗血清，并将同一化合物的另一种模拟物固定在CM5传感器芯片上。可以检测到抗体与芯片表面结合，当有DNC时结合被抑制。芯片表面是使用碱和有机溶剂的组合再生的，并且在数百次进样中非常稳定。一个分析周期(样品进样、芯片再生和系统清洗)需要7 min就能完成。同时，他们介绍了生物传感器技术与LC-MS的对比研究结果。两种方法对于DNC含量的测定显示出良好的一致性，该方法可快速去除阴性样品，确认阳性样品，而LC-MS分析所需的时间更长。该程序具有良好的选择性和灵敏度，可检测出远低于既定的MRL或差动限制(differential action limit, DAL)水平的家禽肝和鸡蛋中的尼卡巴嗪残留标记物DNC。Hu等[63]首次开发了一种基于AuNPs/Zn/Ni-ZIF-8-800 @ 石墨烯复合材料的高效、灵敏的电化学免疫传感器，用于检测牛奶中的莫能菌素。AuNP具有良好的兼容性，可用于固定抗莫能菌素的单克隆抗体，并起到放大信号的作用。这种新型的信号放大免疫传感器检测方法具有高灵敏度和优异的选择性，与其他药物无明显的交叉反应，但该方法并没有应用到实际样本的检测。详见表 5。
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表 5 生物传感器分析技术检测抗球虫药物残留的应用 Table 5 Application of biosensor assay in detection of residues of anticoccidial drugs |
抗球虫药物的种类繁多，且养殖场中常作为饲料添加剂使用，长期食用含有低剂量抗球虫药的动物性食品会危害人类的健康，建立灵敏度高、成本低、操作简便、可同时检测的快速检测方法成为发展趋势。抗球虫药物的样品前处理过程一般选用乙腈或甲醇进行液-液萃取。免疫检测方法操作简便，方法设计较为灵活，通过不同的信号放大系统，如荧光信号，能量变化，分子量大小的改变，这些都能使仪器检测到药物信号从而对样品进行快速的高通量筛选。如今适用性高，应用广泛的免疫检测技术为传统的ELISA检测方法和试纸条。对于需要进行生物信号转换的免疫检测方法，其灵敏度以及选择性高于传统的检测方法，但在实际样品检测过程中有一定的限制，并且很少能够广泛应用于畜牧养殖场中。目前，各种免疫检测技术都要依赖于抗体的特性，性能优异的抗体对免疫检测技术的效果起到了关键性作用，而如今同时检测多种抗球虫药物残留的快速检测方法报道较少。因此，可以将以下几个方面作为研究重点：1)建立非均相体系的免疫检测方法，实现多残留检测。2)通过基因重组技术设计可识别多种药物的抗体。3)设计半抗原并合成抗原，以得到特异性强、灵敏度高的抗体。
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