Diisocyanates are very reactive low molecular weight chemicals that are widely used in the manufacture of polyurethane products. Diisocyanate exposure is one of the most commonly reported causes of occupational asthma. Although diisocyanates have been identified as causative agents of respiratory diseases, the specific mechanisms by which these diseases occur remain largely unknown. Tandem mass spectrometry was used to unambiguously identify the binding site of isocyanates within four model peptides (Leu-enkephalin (Leu-enk, YGGFL), Angiotensin I (DRVYIHPFHL), Substance P-amide (RPKPQQFFGLM-NH2), and Fibronectin-adhesion promoting peptide (FAPP, WQPPRARI)). In each case, isocyanates were observed to react to the N-terminus of the peptide. No evidence of side chain/isocyanate adduct formation exclusive of the N-terminus was observed. However, significant intra-molecular diisocyanate crosslinking between the N-terminal amine and a side chain amine group was observed for arginine, when located within two residues of the N-terminus. Addition of multiple isocyanates to the peptide occurs via polymerization at the N-terminus, rather than addition of multiple isocyanate molecules to varied residues within the peptide. Toluene diisocyanate (TDI)-specific monoclonal antibodies (mAbs) with potential use in immunoassays for exposure and biomarker assessments were produced. A total of 59 unique mAbs were produced (29 IgG1, 14 IgG2a, 4 IgG2b, 2 IgG3 and 10 IgM) against 2,4 and 2,6 TDI bound protein. The reactivities of these mAbs were characterized by a solid phase indirect enzyme-linked immunosorbent assay (ELISA), Dot ELISA and Western immunoblot against various monoisocyanate, diisocyanate and dithioisocyanate protein conjugates. A subset of the mAbs were specific for 2,4 or 2,6 TDI-conjugated proteins only while others reacted to multiple dNCO conjugates including methylene diphenyl diisocyanate- and hexamethelene diisocyanate- human serum albumin . 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. In general, 2,4/2,6 TDI reactive mAbs displayed (1) stronger recognition of monoisocyanate haptenated proteins when the isocyanate was in the ortho position relative to the tolyl group, and were able to discriminate between (2) isocyanate and isothiocyanate conjugates (i.e. between the urea and thiourea linkage); and (3) between aromatic and aliphatic diisocyanates. The mAbs produced were not carrier protein specific with estimated affinity constants toward toluene diisocyanate conjugated human serum albumin ranging from 2.21 x 107 to 1.07 x 1010 M-1 for IgG mAbs. Studies using TDI vapor exposed lung and epithelial cell lines suggest potential utility of these mAbs for both research and biomonitoring of isocyanate exposure.

Diisocyanates (dNCO) such as methylene diphenyl diisocyanate (MDI) are used primarily as cross-linking agents in the production of polyurethane products such as paints, elastomers, coatings and adhesives, and are the most frequently reported cause of chemically induced immunologic sensitization and occupational asthma (OA). Immune mediated hypersensitivity reactions to dNCOs include allergic rhinitis, asthma, hypersensitivity pneumonitis and allergic contact dermatitis. There is currently no simple diagnosis for the identification of dNCO asthma due to the variability of symptoms and uncertainty regarding the underlying mechanisms. Immunological sensitization due to dNCO exposure is traditionally thought to require initial conjugation of the dNCO to endogenous proteins to generate neoantigens, which trigger production of dNCO specific T lymphocytes and ultimately dNCO specific IgE. Testing for dNCO-specific IgE, for diagnosis of dNCO asthma is however, only specific (96-98%) but not sensitive (18-27%). The low prevalence of detectable dNCO specific IgE has been attributed to both assay limitations and a potential IgE-independent dNCO asthma mechanism(s). The identity of the conjugated proteins responsible for the sensitization also remains unknown. It is also not clear whether dNCOs bind to extracellular, cell membrane, or intracellular proteins as a way of triggering non-IgE asthma. Standardization and optimization of immunoassays used to screen for dNCO specific antibodies in sera is important if its utility as a dNCO asthma diagnostic tool is to be achieved. This will potentially improve sensitivity and allow comparison of results across studies. Current studies on assays of dNCO-specific IgE and IgG lack or have limited characterization of the conjugates used. Diisocyanates bound to hemoglobin (Hb), human serum albumin (HSA), and THP-1 proteins were quantified by HPLC with fluorescence detection. Proteomic tandem mass spectrometry (MS) was used to delineate TDI and MDI specific amino acid binding sites on Hb as well as identification of proteins from MDI exposed THP-1 cells. The trinitrobenzene sulfonic acid assay (TNBS) and SDS gel electrophoresis were used to evaluate extent of intra and intermolecular cross-linking in dNCO-HSA conjugates. Binding of monoclonal antibodies (mAbs) to dNCO bound proteins in enzyme-linked immunosorbent assay (ELISA) was used to evaluate antigenicity of dNCO-protein conjugates. The amount of dNCO binding to HSA and Hb increased with the concentration of the dNCO used for conjugation. All the dNCOs reacted with HSA more than with Hb. Eight binding sites were observed with both MDI and TDI on Hb. The N-terminal valines of both the alpha and beta subunits on Hb, lysine 40 of the alpha subunit and lysine 61 of the beta subunit were common binding sites for both TDI and MDI. Lysine 7 of the alpha subunit and lysines 8, 65 and 66 of the beta subunit were unique to MDI. On the other hand, lysines 11, and 16 of the alpha subunit and lysines 17 and 144 of the beta subunit were unique to TDI. Protein bound MDI was detected in a dose-dependent manner in membrane and cytoplasm fractions of MDI exposed THP-1 cells. MDI was also detected in 11 of the 13 cytoplasmic protein bands. The extent of MDI intracellular protein binding was not affected by cytochalasin D, a chemical that binds actin filaments and inhibits active uptake into cells. The extent of cross-linking shown using the TNBS assay was found to increase with amount of dNCO used. Clear bands from both intra and intermolecular cross-linking were observed on all dNCO-Hb/HSA SDS gels. Using ELISA, both TDI-Hb and TDI-HSA conjugates were reactive to monoclonal antibodies produced against TDI conjugated HSA indicating that dNCO-Hb is also antigenic. The best characterization of dNCO-protein conjugates is achieved by the quantitative determination of conjugated dNCO per mole of protein as well as determining the extent of dNCO cross-linking. Although HSA is more reactive to dNCOs than other serum proteins such as Hb, contribution from other serum proteins to development of OA should not be overlooked as dNCO-Hb was found to be reactive to dNCO specific mAbs. dNCO-conjugated proteins identified in the soluble fraction of MDI exposed THP-1 cells were all of intracellular origin suggesting that MDI can cross the cell membrane and react with intracellular proteins. The entry of MDI into live cells is a passive process, as the extent of intracellular binding was not affected by cytochalasin D. The present study support the potential involvement of dNCO-haptenated membrane and intracellular proteins in development of non-IgE dNCO asthma.