Broadening the Detection Spectrum of Small Analytes Using a Two-Antibody-Designed Hybrid Immunoassay

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.