Franek Milan, Diblikova Iva, Cernoch Ivo, Vass Maria, Hruska Karel
Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic.
Anal Chem. 2006 Mar 1;78(5):1559-67. doi: 10.1021/ac0514422.
Development of antibodies with broad specificity recognition for sulfonamide drugs was found to be surprisingly difficult when conventional immunochemical strategies were applied to hapten design. To improve the cross-reactivity pattern of antibodies for the family of sulfonamide drugs, a novel strategy based on the single-ring (fragment-derived) hapten moieties with different spacer substituent lengths was employed for the preparation of immunogens, coating conjugates, and enzyme competitors. The rabbit antibodies raised against a common (one-ring) p-aminobenzenesulfonamide hapten moiety (attached to a carrier protein through the N-1 position) in combination with a homologous hapten-peroxidase tracer allowed the detection of 15 sulfonamide species at the maximum residue limit level using direct ELISA. The two-ring 6-(4-aminobenzensulfonylamino)hexanoic hapten mimics, previously reported in the literature as a weak generic antigen, generated surprisingly superior immune responses in rabbits. The antibodies raised against this two-ring hapten were capable of detecting at least 19 and 17 sulfonamides in a direct ELISA system at the regulatory level with sensitivities corresponding to 20 and 50% binding inhibition, respectively. A negligible cross-reaction with N4 metabolites makes it possible to measure responses of parent sulfonamides in the presence of their metabolized forms. In skimmed milk, the highest limit of detection (LOD) for sulfacetamide defined as 20% inhibition was 65.2 microg x L(-1) (IC20 value), whereas the additional 18 sulfonamides tested exhibited LODs in the range of 0.2-36.8 microg x L(-1). This sensitivity allows simple multisulfonamide tests to be established for use in the laboratory or on site.
当将传统免疫化学策略应用于半抗原设计时，发现开发对磺胺类药物具有广泛特异性识别的抗体出奇地困难。为了改善抗体对磺胺类药物家族的交叉反应模式，采用了一种基于具有不同间隔取代基长度的单环（片段衍生）半抗原部分的新策略来制备免疫原、包被偶联物和酶竞争剂。针对常见的（单环）对氨基苯磺酰胺半抗原部分（通过N-1位连接到载体蛋白）产生的兔抗体，与同源半抗原-过氧化物酶示踪剂结合，使用直接ELISA法在最大残留限量水平可检测15种磺胺类物质。文献中先前报道的作为弱通用抗原的双环6-(4-氨基苯磺酰氨基)己酸半抗原模拟物，在兔子中产生了出奇优异的免疫反应。针对这种双环半抗原产生的抗体在直接ELISA系统中能够在监管水平检测至少19种和17种磺胺类药物，灵敏度分别对应于20%和50%的结合抑制。与N4代谢物的交叉反应可忽略不计，这使得在存在其代谢形式的情况下能够测量母体磺胺类药物的反应。在脱脂牛奶中，定义为20%抑制的磺胺醋酰的最高检测限（LOD）为65.2μg x L(-1)（IC20值），而测试的另外18种磺胺类药物的LOD在0.2 - 36.8μg x L(-1)范围内。这种灵敏度使得能够建立简单的多磺胺类药物检测方法，用于实验室或现场。
