Cliquet Patricia, Cox Eric, Haasnoot Willem, Schacht Etienne, Goddeeris Bruno Marie
Laboratory of Veterinary Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.
J Agric Food Chem. 2003 Sep 24;51(20):5835-42. doi: 10.1021/jf034316c.
To develop a sulfonamide-specific ELISA, different attempts were made to obtain monoclonal antibodies specific for the common structure of sulfonamides. In a first approach, sulfanilamide was linked to albumins using glutaraldehyde or a succinimide ester as cross-linker. A weak immune response or none at all was induced after immunization of mice with those conjugates. High antibody titers were obtained with conjugates where sulfanilamide was linked to albumins or casein (azocasein) with a diazotation reaction. However, the antibodies were only highly specific for the bound sulfanilamide molecule. In a second approach, sulfonamide-protein conjugates were used in which the sulfonamide molecule is linked at its side chain, leaving the common structure of sulfonamides unchanged. Three sulfonamide derivatives (S, TS, and PS, previously described in the literature) containing a carboxyl group in their side chain were linked to proteins using a carbodiimide mediated reaction. Immunization with the S-conjugates led to high antibody titers, but the antibodies were only highly specific for the bound S-molecule. Group-specific antibodies were obtained after immunization with the PS- and TS-conjugates. It was described that immunization with PS-conjugates lead to the recognition of other sulfonamides (sulfamethazine, -merazine, -diazine, and -dimethoxine) that are not well recognized by antibodies induced after immunization with TS-conjugates. Therefore, we tried to guide the immune response in the direction of recognition of the common structure of sulfonamides by immunizing the animals alternately with PS- and TS-conjugates. The polyclonal antibodies of the mice indeed had a broader specificity, but the specificity of the monoclonals obtained after fusion experiments was not influenced. Immunization with TS-conjugates seemed sufficient to obtain sulfonamide-specific monoclonal antibodies. With the best monoclonal (mAb 3B5B10E3) two competitive inhibition (ci) ELISA's were developed: one coated with antigen and the other coated with the monoclonal antibody. Sulfadiazine, -dimethoxine, -thiazole, -pyridine, and -methoxazole were detected in both ELISA's at their MRL-value (100 ppb) in buffer solution. Sulfadiazine, sulfathiazole, and sulfamethoxazole could even be detected at 10 ppb.
为开发一种磺胺类药物特异性酶联免疫吸附测定法（ELISA），人们进行了不同尝试以获得对磺胺类药物共同结构具有特异性的单克隆抗体。在第一种方法中，使用戊二醛或琥珀酰亚胺酯作为交联剂将磺胺与白蛋白连接。用这些缀合物免疫小鼠后诱导出的免疫反应较弱或根本没有。用重氮化反应将磺胺与白蛋白或酪蛋白（偶氮酪蛋白）连接的缀合物可获得高抗体滴度。然而，这些抗体仅对结合的磺胺分子具有高度特异性。在第二种方法中，使用了磺胺 - 蛋白质缀合物，其中磺胺分子在其侧链处连接，磺胺类药物的共同结构保持不变。三种在其侧链中含有羧基的磺胺衍生物（S、TS和PS，先前在文献中有所描述）通过碳二亚胺介导的反应与蛋白质连接。用S - 缀合物免疫导致高抗体滴度，但这些抗体仅对结合的S分子具有高度特异性。用PS - 和TS - 缀合物免疫后获得了组特异性抗体。据描述，用PS - 缀合物免疫会导致对其他磺胺类药物（磺胺二甲嘧啶、磺胺间甲氧嘧啶、磺胺嘧啶和磺胺二甲氧嘧啶）的识别，而用TS - 缀合物免疫诱导产生的抗体对这些药物识别效果不佳。因此，我们尝试通过用PS - 和TS - 缀合物交替免疫动物来引导免疫反应朝着识别磺胺类药物共同结构的方向发展。小鼠的多克隆抗体确实具有更广泛的特异性，但融合实验后获得的单克隆抗体的特异性并未受到影响。用TS - 缀合物免疫似乎足以获得磺胺类药物特异性单克隆抗体。利用最佳的单克隆抗体（mAb 3B5B10E3）开发了两种竞争性抑制（ci）ELISA：一种包被抗原，另一种包被单克隆抗体。在两种ELISA中均检测到缓冲溶液中磺胺嘧啶、磺胺二甲氧嘧啶、磺胺噻唑、磺胺吡啶和磺胺甲恶唑的最高残留限量值（100 ppb）。磺胺嘧啶、磺胺噻唑和磺胺甲恶唑甚至在10 ppb时也能被检测到。
