Draisci R, delli Quadri F, Achene L, Volpe G, Palleschi L, Palleschi G
Laboratorio Medicina Veterinaria, Istituto Superiore di Sanità, Rome, Italy.
Analyst. 2001 Nov;126(11):1942-6. doi: 10.1039/b104939a.
A new sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of two macrolides (erythromycin and tylosin) in bovine muscle was developed, using the mouse monoclonal antibodies anti-erythromycin and anti-tylosin. The competitive indirect assay was performed using an erythromycin (or tylosin)-BSA conjugate as a coating molecule; after competition between free and coated analytes for the antibodies, the activity of the horseradish peroxidase-labelled antiglobulins was measured electrochemically using 3,3',5,5'-tetramethylbenzidine (TMB) as substrate. The detection limit of the assay was 0.4 ng ml(-1) for erythromycin and 4.0 ng ml(-1) for tylosin, while the sensitivity (25% inhibition concentration) was 1.4 ng ml(-1) for erythromycin and 13.0 ng ml(-1) for tylosin. The specificity of the assay was assessed by studying the cross-reactivity of various macrolides other than erythromycin and tylosin. The results indicate that the monoclonal antibodies anti-erythromycin and anti-tylosin can readily distinguish the target compound from other macrolides, with the exception of roxithromycin, a semisynthetic macrolide antibiotic derived from erythromycin. Fortified and real samples were analysed by the developed ELISA method and results confirmed by micro-LC-MS-MS using an atmospheric pressure ionisation (API) source and an ionspray (IS) interface. The latter provides unequivocal identification and quantification of the analytes at the level of interest. The ELISA assay showed precision (RSD) values ranging from 6.3 to 11.4% for erythromycin and from 7.5 to 12.6% for tylosin; the accuracy (relative error, RE) ranged from -16.0 to -9.8% and from -9.5 to 8.0% for erythromycin and tylosin, respectively. All results obtained demonstrate that the electrochemical ELISA is a suitable method for a sensitive, simple, rapid and reliable screening of the two macrolides in animal tissues.
利用抗红霉素和抗泰乐菌素的小鼠单克隆抗体，开发了一种用于检测牛肌肉中两种大环内酯类药物（红霉素和泰乐菌素）的新型灵敏电化学酶联免疫吸附测定法（ELISA）。竞争性间接测定法使用红霉素（或泰乐菌素）-牛血清白蛋白偶联物作为包被分子；游离和包被的分析物竞争抗体后，使用3,3',5,5'-四甲基联苯胺（TMB）作为底物，通过电化学法测定辣根过氧化物酶标记的抗球蛋白的活性。该测定法对红霉素的检测限为0.4 ng ml⁻¹，对泰乐菌素为4.0 ng ml⁻¹，而灵敏度（25%抑制浓度）对红霉素为1.4 ng ml⁻¹，对泰乐菌素为13.0 ng ml⁻¹。通过研究红霉素和泰乐菌素以外的各种大环内酯类药物的交叉反应性来评估该测定法的特异性。结果表明，抗红霉素和抗泰乐菌素的单克隆抗体能够很容易地将目标化合物与其他大环内酯类药物区分开来，但来源于红霉素的半合成大环内酯类抗生素罗红霉素除外。通过开发的ELISA方法分析了加标样品和实际样品，并使用大气压电离（API）源和离子喷雾（IS）接口的微液相色谱-质谱-质谱法（micro-LC-MS-MS）对结果进行了确认。后者能够在感兴趣的水平上对分析物进行明确的鉴定和定量。ELISA测定法中，红霉素的精密度（相对标准偏差，RSD）值范围为6.3%至11.4%，泰乐菌素为7.5%至12.6%；准确度（相对误差，RE）对红霉素分别为-16.0%至-9.8%，对泰乐菌素为-9.5%至8.0%。获得的所有结果表明，电化学ELISA是一种适用于灵敏、简单、快速且可靠地筛选动物组织中这两种大环内酯类药物的方法。
