Characterization of methylene diphenyl diisocyanate-haptenated human serum albumin and hemoglobin

Abstract

Protein haptenation by polyurethane industrial intermediate methylene diphenyl diisocyanate (MDI) is thought to be an important step in the development of diisocyanate (dNCO)-specific allergic sensitization; however, MDI haptenated albumins used to screen specific antibody are often poorly characterized. Recently, the need to develop standardized immunoassays using a consistent, well characterized dNCO-haptenated protein to screen for the presence of MDI-specific IgE and IgG from workers’ sera has been emphasized and recognized. This has been challenging to achieve due to the bivalent, electrophilic nature of dNCO leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the present study, MDI was reacted with human serum albumin (HSA) and hemoglobin (Hb) at molar ratios ranging from 1:1 to 40:1 MDI: protein. Adducts were characterized by (1) loss of available trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (2) electrophoretic migration in polyacrylamide gels, (3) quantification of methylene diphenyl diamine following acid hydrolysis and (4) immunoassay. Concentration dependent changes in all the above noted parameters were observed demonstrating increase in both number and complexity of conjugates formed with increasing MDI concentration. In conclusion, a series of bio-analytical assays should be performed to standardize MDI-antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers which in total indicate degree of intra- and inter-molecular cross-linking, number of dNCO bound, number of different specific binding sites on the protein and degree of immuno-reactivity.

Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin

“…Examination of the tandem mass spectra of the tryptic peptides allowed assignment of conjugation sites on Hb as previously described 32 . Hb has 2 alpha and 2 beta subunits and mass spectrometry allowed identification of the parent subunit from which each binding site originated.…”

“…Table 3 shows a concentration-dependent loss of available primary amines with increasing TDI and HDI concentrations and, thus, an increase in the amount of dNCO cross-linking of protein residues 32 . At TDI and HDI concentrations ranging from 1:1 to 10:1, the degree of cross linking is not statistically different between the 2 diisocyanates.…”

“…TDI–protein adducts were prepared as described previously for MDI-HSA/Hb conjugates 32 . Briefly, 0.5 mg/ml protein solutions were prepared in 0.01 M PBS (pH 7.4).…”

“…Our previous work focused on characterization of methylene diphenyl diisocyanate (MDI)-HSA and MDI-Hb conjugates 32 . Although HSA is the most common carrier protein used for dNCO antibody immunoassays 21,22 , other proteins, however, have also been found to be potentially modified by dNCO exposure 22–25,33,34 but the immunogenicity of adducted proteins other than albumins has not been reported.…”

“…In the present study we extend the characterization of the dNCO-protein conjugates from MDI to understand reactivity differences among dNCOs such as TDI and HDI that can impact assay sensitivities and standardization protocols. Quantification of the amount of TDI and HDI bound per mole protein was conducted by analyses of the corresponding hydrolysis products following assay hydrolysis of the conjugate, derivitization of the diamines and HPLC florescence detection 32 . Cross linking was evaluated using the 2, 4, 6-trinitrobenzene sulphonic acid (TNBS) assay, which is a primary amine-specific spectrophotometric probe.…”