Characterization of methylene diphenyl diisocyanate-haptenated human serum albumin and hemoglobin.
Mhike-M; Chipinda-I; Hettick-JM; Simoyi-RH; Lemmons-A; Green-BJ; Siegel-PD
Anal Biochem 2013 Sep; 440(2):197-204
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.
Proteins; Allergens; Allergies; Chemical-properties; Chemical-reactions; Workers; Work-environment; Humans; Men; Women; Respiratory-system-disorders; Bronchial-asthma; Pulmonary-system-disorders;
Author Keywords: Occupational Asthma; Immunoassay standardization; Human serum albumin; Hemoglobin; Methylene diphenyl diisocyanate
Paul D. Siegel, Ph.D., HELD/NIOSH/CDC, 1095 Willowdale Rd., Morgantown WV 26505-2888
Healthcare and Social Assistance