Recombinant Expression and Engineering of MDI- and HDI-Specific Monoclonal Antibodies for Proof-of-Concept Detection...

Methylene-diphenyl diisocyanate (MDI) and hexamethylene diisocyanate (HDI), important chemicals for polyurethane production, can react with self-molecules, trigger immune responses, and cause asthma. To validate molecular determinants of diisocyanate immune recognition and develop renewable immunoassay reagents, we generated MDI- and HDI-specific recombinant monoclonal antibodies (mAbs) from hybridoma-derived variable-region DNA sequences using a human embryonic kidney (HEK) cell line expression system. Three anti-MDI and one anti-HDI mouse IgG1/κ mAbs were produced, purified, and evaluated for binding to MDI, HDI, and control antigens, including toluene diisocyanate (TDI) and mock-reacted protein. To support assay standardization in research and translational applications, we engineered one anti-MDI (DA5) and one anti-HDI (AG5) mAb as human Fc chimeras (human IgG1 or IgE heavy-chain constant regions with human κ light-chain constant region) and confirmed that chimerization preserved antigen specificity. As a proof-of-principle, one of the chimeric mAbs was evaluated as a candidate calibrator in an enzyme-linked immunosorbant assay (ELISA) format to quantify diisocyanate-specific antibodies in serum from an MDI-exposed worker. In addition, mAb DA5 was engineered with a FLAG epitope to enable immunoassays for detecting MDI-protein reaction products in airway fluid, including sandwich ELISA (with anti-MDI mAb CE2) and Western blot formats. Together, these data show that diisocyanate specificity can be recapitulated by recombinant expression from deposited variable-region DNA sequences. The engineered reagents enable immunoassay detection of MDI-protein adducts in complex matrices and serve as proof-of-concept calibrators for ELISA-based quantification of anti-diisocyanate antibodies in human serum.