The administration of xenogenic monoclonal antibodies (MAbs) leads in many cases to a host immune response represented by the generation of antibodies that can be directed against allotypic, isotypic, and idiotypic determinants present on the xenogeneic MAb. Anti-idiotypic antibodies (Ab2s) can be detected by measuring their specific reactivity in sandwich assays using their ability to cross-link labeled Abl to Abl attached to a solid phase; however, when the MAb used for these studies reacts with a multideterminant tumor-associated antigen found in the circulation (e.g., TAG-72), the utility of these anti-idiotype assays may be limited. To determine the levels of anti-idiotypic antibodies that could be detected in patients undergoing MAb B72.3-based immunodiagnostic and immunotherapeutic protocols, we investigated the ability of a solid-phase sandwich radioimmunoassay (RIA) to detect anti-idiotypic antibodies raised against 872.3. Furthermore, to overcome the interference of circulating TAG-72 andlor antibodies to allotypic and isotypic determinants in the evaluation of an anti-idiotypic response, we developed a methodology using CC49-coated resin to adsorb the interfering molecules (CC49 is a second anti-TAG-72 MAb). Under the conditions established, all of the TAG-72 antigen was removed by adsorption with MAb CC49. Furthermore, since the treatment used an isotype-identical murine MAb, the binding due to the anti-mouse antibodies, other than the anti-idiotype, was completely abolished after a treatment with MAb CC49, leaving only the anti-idiotype component. Analysis of serum samples from patients who had received 872.3 that were positive for human anti-mouse antibodies in the 872.3 sandwich RIA, after the adsorption with CC49 resin, revealed the presence of a 872.3binding component in 2 of 12 samples. The ability of the adsorbed sera to compete with an anti-B72.3-idiotype MAb for the binding sites present on the MAb 872.3 confirmed the anti-idiotypic nature of the component being detected in the patient sera.