Toluene diisocyanate is a high production chemical with major uses in the production of flexible polyurethane foams, elastomers, and coatings. Exposure to diisocyanates can occur in both polyurethane production and during the use of the diisocyanate containing products by the consumer. Both aromatic and aliphatic diisocyanates are etiological agents of hypersensitivity diseases including asthma, rhinitis, allergic contact dermatitis, and hypersensitivity pneumonitis.(16,19–21)
conjugation of TDI to proteins in the human airways is thought to be a primary event in TDI exposure.(4)
However, the characterization of dNCO haptenated proteins is complicated by the ability of the dNCOs to haptenate multiple proteins to self-polymerize and form both intra- and intermolecular cross-links with diverse proteins and non-protein species.
The antigens used in the characterization of the MAbs produced in the present study can be divided according to (1) the chemical nature of the carrier protein as basic (lysozyme), acidic (HSA), or insoluble (keratin); (2) the origin of the carrier protein as animal or human; and (3) the chemical nature of the isocyanate adduct. The TDI-adduct formation was modified by changing the position of the NCO group on the benzene ring, removing one NCO, substituting one NCO with a methyl group, or substituting dNCO groups with dNCS groups.
The isocyanate functional groups in TDI can potentially react with a hydroxyl group (in hydrophobic pockets) to form a urethane linkage, a thiol group to form a thiourea, or an amine group to form a urea. 2,4 TDI is an asymmetrical molecule and thus has two isocyanate groups of different reactivity. The 4 position is more reactive than the 2 position because it is more accessible. 2,6 TDI is a symmetrical molecule and thus has two isocyanate groups of similar reactivity, similar to the 2 position of 2,4 TDI. Reaction of one isocyanate group will cause a change in the reactivity of the second isocyanate group since both isocyanate groups are attached to the same aromatic ring. The first NCO of a diisocyanate may form a urea linkage with a primary amine of a protein while the second NCO may be hydrolyzed to an amine with the potential to react with additional NCO groups to undergo intra- or intermolecular cross-linking with other proteins resulting in dimers, trimers, and so forth.(12)
Intra- and intermolecular crossing can also be mediated by a single dNCO molecule reacting to two separate amino acids. All MAbs produced herein reacted with proteins that contained intra- and intermolecular TDI-mediated cross-linking, suggesting that their reactivity was not impaired by the presence of cross-linking.
Examination of the MAbs reactivity towards monoisocyanate-haptenated proteins was used to assess the need of molecular cross-linking on the ability of the MAbs to recognize TDI-haptenated proteins. OTI and PTI are monoisocyanates with the second isocyanate group removed from the ortho and para positions relative to the methyl group. 2,3 DMPI, 2,5 DMPI, and 3,5 DMPI are monoisocyanates with the ortho and para positions substituted with methyl groups. PI has only one isocyanate and no methyl group attached to the phenyl ring. All monoclonal antibodies recognized monoisocyanate haptenated albumin, suggesting that the cross-linking was not critical to immune recognition. The assay format, however, was found to influence the MAb reactivity with the various haptenated species. Having an isocyanate or a substitution at the ortho position on the tolyl group was critical to MAb recognition in the ELISA format. This was true for all three MAbs, suggesting a possible influence of ELISA plate chemistry in the interactions between the MAbs and haptenized protein. The MAbs recognized haptenated albumin in which there was substitution of one isocyanate on the tolyl group (in the Western blot and dot blot formats) but not simple removal of the isocyanate. Interestingly, higher molecular weight proteins than ~66.5
kDa in the stock HSA as observed in the protein blot were also haptenated by TDI and recognized by the MAbs ( and ). These MAb recognized MDI- and/or HDI-conjugated HSA, in addition to the TDI-HSA. This suggests that the resultant bond formation following reaction of the dNCO to protein was important in the recognition of TDI-haptenated proteins by the MAbs.
The reactivity of the antibodies may also be influenced by the choice of the experimental carrier protein. The MAbs reacted with 2,4 TDI bound to HSA or MSA and also to KLH, which is a non-mammalian protein, indicating the importance of the dNCO-protein bond rather than the source species of the carrier protein. Although lysozyme and keratin were demonstrated by matrix-assisted laser desorption ionization and/or loss of primary amines (data not shown) to be haptenated, none of the MAbs in the present study showed reactivity toward their conjugates. This may be due to poor binding of the conjugates to the ELISA plate surface, lower haptenation rates, or steric inaccessibility of the TDI group on these carrier proteins.
A total of 10 MAbs (all IgMs) generated from TDI vapor-exposed mice were isolated during the initial screening against TDI-HSA; however, only three MAbs showed no or very low reactivity to unconjugated HSA. The reason for the high reactivity of the other MAbs with HSA is not known but we were unable to block this binding with either milk or BSA. It is possible that haptenation of proteins may also lead to recognition of the self protein by the immune system as seen with exposure to aldehydes(22)
although this has not been investigated for isocyanate exposures. Interestingly, these TDI vapor-sensitized mice had IgG anti-2,4 TDI-MSA titers of 18,106
7,994 (sera dilution to reach an ELISA OD >0.1). IgM anti-2,4 TDI-MSA was not assessed in these mice; however, anti-2,4 TDI-MSA IgM was not observed in the sera of another group of mice exposed to an identical inhalation protocol (data not shown).
Future potential applications of these MAbs include the isolation of endogenous carrier proteins that are conjugated after natural or experimental exposure episodes. The purified proteins can then be identified and their structure and chemical linkages determined by mass spectrometry. We recently demonstrated the potential for such methods for isocyanate-peptide adducts using tandem mass spectrometry.(23)
The MAbs may also be useful reagents for the development of immunoassays designed for dNCO exposure assessments.
In summary, we have successfully developed monoclonal antibodies that recognize dNCO protein adducts from TDI vapor-exposed mice. The specificity of these antibodies was demonstrated by ELISA, Western blot, and dot blot analyses. The antibodies may be useful tools for the identification of endogenous dNCO-modified proteins and the characterization of the importance of the type of chemical linkage in terms of isocyanate toxicity.