University of Macau / Institute of Chinese Medical Sciences

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.

This study focuses on the utilization of metal-dependent monoclonal antibodies for large-scale chromatography and addresses an aspect that has been cited to lower immunosorbent performance, namely "orientation" of antibodies on matrices. The antibodies used in this study, the "EDTAdependent" 7D7BlO and the "Ca²⁺ -dependent" HPC4 are directed against human Protein C (PC). The 7D7BI0 antibody was characterized in terms of its metaldependency and specificity. The region of PC (epitope) recognized by 7D7BlO was identified as the first 15 residues in the NH₂-terminal. Immunosorbents made with 7D7BI0 provided highly pure and functional PC. The "orientation" of the antibodies on matrices was addressed in two ways. In the first approach, performance of immunosorbents coupled through carbohydrate moieties were compared with immunosorbents coupled through peptide regions. Coupling via carbohydrate linkages, which is generally believed to be Fc-directed, did not have any advantage in terms of efficiency and recovery over coupling via peptide. / Ph. D.