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

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• 作者：Morgen Mhike ; Itai Chipinda ; Justin M. Hettick ; Reuben H. Simoyi ; Angela Lemons ; Brett J. Green ; Paul D. Siegel
• 关键词：Occupational asthma ; Immunoassay standardization ; Human serum albumin ; Hemoglobin ; Methylene diphenyl diisocyanate
• 刊名：Analytical Biochemistry
• 出版年：2013
• 出版时间：15 September, 2013
• 年：2013
• 卷：440
• 期：2
• 页码：197-204
• 全文大小：712 K

文摘

Protein haptenation by polyurethane industrial intermediate 4,4¡ä-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 dNCOs, leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the current 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 (i) loss of available 2,4,6-trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (ii) electrophoretic migration in polyacrylamide gels, (iii) quantification of methylene diphenyl diamine following acid hydrolysis, and (iv) immunoassay. Concentration-dependent changes in all of the above noted parameters were observed, demonstrating increases in both number and complexity of conjugates formed with increasing MDI concentrations. In conclusion, a series of bioanalytical assays should be performed to standardize MDI-antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers that in total indicate degree of intra- and intermolecular cross-linking, number of dNCOs bound, number of different specific binding sites on the protein, and degree of immunoreactivity.