The recognition spectrum of immunoassays developed on the basis of class-specific antibodies can include the several nearest analytes but rarely all of the desired representatives of the group. The situation may be sufficiently improved using a hybrid assay combining two antibodies with specificities that complement each other. Two monoclonal antibodies (mAb) with broad but different specificities toward sulfonamides were examined for their binding to a panel of hapten conjugates. mAb–hapten pairs without mutual cross-reactions were identified, and classical direct antigen-coated and mAb-coated ELISAs were developed as formats with referent specificities. Both interactions were combined in a single hybrid assay, which was designed as a one-step double-competitive sandwich-ELISA. For this assay, the intermediate bifunctional reagent mAb(1)-hapten(2) conjugate was synthesized and able to simultaneously bind to hapten(1) and be bound by mAb(2). Formation of a two-mAbs sandwich complex was inhibited by competitors of interaction(1) as well as by competitors of interaction(2). Thus, due to the summation effect, simultaneous determination of analytes recognized by both mAbs was achieved. The hybrid assay can be performed in two reversed arrangements using a coating antigen or coating antibody, the characteristics of which were compared and found to be similar in sensitivity and extended specificity. The suitability of the developed test for the determination of 14 sulfonamides at their maximum residue limit (MRL) concentration was demonstrated using the examples of turkey muscle and milk samples.

中文翻译：

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使用两抗体设计的混合免疫测定法拓宽小分析物的检测谱

基于类别特异性抗体开发的免疫分析方法的识别范围可以包括几种最接近的分析物，但很少包括该组的所有所需代表。使用结合两种具有互补性的特异性的抗体的杂交试验，可以充分改善这种情况。检查了两种对磺酰胺类具有广泛但不同特异性的单克隆抗体（mAb）与半抗原缀合物的结合。鉴定出没有相互交叉反应的mAb-半抗原对，并开发了具有抗原特异性的经典直接抗原包被和mAb包被的ELISA。两种相互作用都在一个杂交试验中结合在一起，该杂交试验设计为一步双竞争夹心ELISA。对于此测定，合成了中间双功能试剂mAb（1）-hapten（2）偶联物，该偶联物能够同时与半抗原（1）结合并被mAb（2）结合。相互作用的竞争者（1）和相互作用的竞争者（2）抑制了两个mAbs三明治复合物的形成。因此，由于加和效应，可以同时测定两个mAb识别的分析物。杂交试验可以使用包被抗原或包被抗体以两种相反的方式进行，比较了它们的特征，发现其敏感性和扩展特异性相似。以火鸡肌肉和牛奶样品为例，证明了开发的测试方法适合测定14种磺酰胺类药物的最大残留限量（MRL）浓度。