Structural insights into ternary immunocomplex formation and cross‐reactivity: binding of an anti‐immunocomplex FabB12 to Fab220‐testosterone complex

Anti‐immunocomplex (Anti‐IC) antibodies have been used in developing noncompetitive immunoassays for detecting small molecule analytics (haptens). These antibodies bind specifically to the primary antibody in complex with hapten. Although several anti‐IC antibody–based immunoassays have been developed, structural studies of these systems are very limited. In this study, we determined the crystal structures of anti‐testosterone Fab220 in complex with testosterone and the corresponding anti‐IC antibody FabB12. The structure of the ternary complex of testosterone, Fab220, and FabB12 was predicted using LightDock and AlphaFold. The ternary complex has a large (~ 1100 Å2) interface between antibodies. The A‐ring of the testosterone bound by Fab220 also participates in the binding of the anti‐IC antibody. The structural analysis was complemented by native mass spectrometry. The affinities for testosterone (TES) and three cross‐reactive steroids [dihydrotestosterone (DHT), androstenedione (A4), and dehydroepiandrosterone sulfate (DHEA‐S)] were measured, and ternary complex formation was studied. The results clearly show the ternary complex formation in the solution. Although DHT showed significant cross‐reactivity, A4 and DHEA‐S exhibited minor cross‐reactivity. Here, we present a comprehensive structural description of how an anti‐immunocomplex antibody binds to the primary antibody in complex with its hapten, testosterone. We utilized X‐ray crystallography, AlphaFold and LightDock structure predictions, and native mass spectrometry to understand the molecular basis of specificity, cross‐reactions, and the ternary complex formation.