Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
Anal Biochem. 2013 Sep 15;440(2):197-204. doi: 10.1016/j.ab.2013.05.022. Epub 2013 Jun 4.
Protein haptenation by polyurethane industrial intermediate 4,4'-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.
聚氨酯工业中间体 4,4'-亚甲基二苯基二异氰酸酯（MDI）与蛋白质的结合被认为是二异氰酸酯（dNCO）特异性过敏致敏发展的重要步骤；然而，用于筛选特异性抗体的 MDI 结合白蛋白通常特征描述较差。最近，人们强调并认识到需要开发使用一致的、特征良好的 dNCO 结合蛋白的标准化免疫测定法，以筛选工人血清中 MDI 特异性 IgE 和 IgG 的存在。由于 dNCO 的二价亲电性，这一直具有挑战性，导致能够产生多种交联的蛋白质物种和蛋白质的聚合添加。在当前的研究中，MDI 与人血清白蛋白（HSA）和血红蛋白（Hb）在摩尔比为 1:1 至 40:1 MDI/蛋白的范围内反应。加合物通过以下方式进行表征：（i）与仲胺结合的 2,4,6-三硝基苯磺酸（TNBS）的可用性损失，（ii）在聚丙烯酰胺凝胶中的电泳迁移，（iii）酸水解后二亚甲基苯基二胺的定量，和（iv）免疫测定。所有上述参数均观察到浓度依赖性变化，表明随着 MDI 浓度的增加，形成的缀合物的数量和复杂性均增加。总之，应该进行一系列生物分析测定，以在批次和实验室之间标准化 MDI 抗原制剂，用于测量暴露于工人中的特异性抗体，这些测定总共表明了分子内和分子间交联的程度、结合的 dNCO 数量、蛋白质上不同特异性结合位点的数量和免疫反应性。
