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An antibody with broad specificity and principally depending on hapten structure and size is a key reagent for developing a class-selective immuno­assay. In the present study, three new generic haptens of antibacterial synergists (ASGs) were proposed using trimetho­prim as the starting molecule. These haptens contained carboxyl groups on the meta position of trimethoxy­benzene for conjugating to protein, while, the common moiety of ASGs, i.e., diamino­pyrimidine, was intentionally and maximally exposed to the immune system in animals in order to induce antibodies with broad specificity against ASGs. Five monoclonal antibodies (mAbs) were finally obtained, and 5C4 from the hapten with a short spacer arm, named Hapten A, showed not only uniform broad specificity but also high affinity to all five ASGs. We further determined the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. An indirect competitive ELISA (icELISA)-based 5C4 was established and exhibited IC50 values of 0.067–0.139 μg L–1 with cross-reactivity of 48.2%–418.7% for the five ASGs in buffer under optimal conditions. The calculated limits of detection of the icELISA for chicken and milk were 0.06–0.8 μg kg–1 and 0.05–0.6 μg L–1, respectively. The recoveries in spiked chicken and milk samples were 75.2%–101.4% with a coefficient of variation less than 14.3%. In summary, we have developed, for the first time, a rapid and reliable icELISA for ASGs with significantly improved sensitivity and class selectivity.

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