Monoclonal Antibodies Against Toluene Diisocyanate Haptenated Proteins from Vapor-Exposed Mice

Abstract

Toluene diisocyanate (TDI) is an industrially important polymer cross-linker used in the production of polyurethane. Workplace exposure to TDI and other diisocyanates is reported to be a leading cause of low molecular weight-induced occupational asthma (OA). Currently we have a limited understanding of the pathogenesis of OA. Monoclonal antibodies (MAbs) that recognize TDI bound proteins would be valuable tools/reagents, both in exposure monitoring and in TDI-induced asthma research. We sought to develop toluene diisocyanate (TDI)-specific MAbs for potential use in the development of standardized immunoassays for exposure and biomarker assessments. Mice were exposed 4 h/day for 12 consecutive weekdays to 50 ppb, 2,4;2,6 TDI vapor (80/20 mixture). Splenocytes were isolated 24 h after the last exposure for hybridoma production. Hybridomas were screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against a 2,4 TDI-human serum albumin (2,4 TDI-HSA) protein conjugate. Three hybridomas producing 2,4 TDI-HSA reactive IgM MAbs were obtained. The properties of these MAbs (isotype and reactivity to various protein-isocyanate conjugate epitopes) were characterized using ELISA, dot blot, and Western blot analyses. Western blot analyses demonstrated that some TDI conjugates form inter- and intra-molecular links, resulting in multimers and a change in the electrophoretic mobility of the conjugate. These antibodies may be useful tools for the isolation of endogenous diisocyanate-modified proteins after natural or experimental exposures and for characterization of the toxicity of specific dNCOs.