Elsevier

Analytical Biochemistry

Volume 400, Issue 2, 15 May 2010, Pages 251-258
Analytical Biochemistry

Antigenic changes in human albumin caused by reactivity with the occupational allergen diphenylmethane diisocyanate

https://doi.org/10.1016/j.ab.2010.01.037Get rights and content

Abstract

Diphenylmethane diisocyanate (MDI), the chemical commonly used as a cross-linking agent in commercial polyurethane production, is a well-recognized cause of asthma. Reaction products between MDI and “self” proteins are hypothesized to act as antigens capable of inducing airway inflammation and asthma; however, such MDI antigens remain incompletely understood. We used a variety of analytical methods to characterize the range of MDI–albumin reaction products that form under physiological conditions. Sites of MDI conjugation on antigenic MDI–albumin products, as defined by serum immunoglobulin G (IgG) from MDI-exposed workers, were determined by high-performance liquid chromatography (HPLC) followed by tandem mass spectrometry (MS/MS). The data identified 14 MDI conjugation sites (12 lysines and 2 asparagines) on human albumin and highlight reaction specificity for the second lysine in dilysine (KK) motifs, and this may be a common characteristic of “immune-sensitizing” chemicals. Several of the MDI conjugation sites are not conserved in albumin from other species, and this may suggest species differences in epitope specificity for self protein (albumin)–isocyanate conjugates. The study also describes new applications of contemporary proteomic methodology for characterizing and standardizing MDI–albumin conjugates destined for use in clinical research.

Section snippets

Chemicals and reagents

Low endotoxin certified human albumin was obtained from Sigma Chemical (St. Louis, MO, USA), 4′4-diphenyl methane diisocyanate was obtained from Aldrich (St. Louis, MO, USA), and acetone was obtained from J.T. Baker (Phillipsburg, NJ, USA).

Reactivity of MDI with human albumin

Human albumin was solubilized in phosphate-buffered saline (PBS: 20 mM phosphate and 140 mM NaCl, pH 7.2) at 0.5% (w/v) to achieve a concentration (∼74 μM) similar to human airway epithelial lining fluid [17]. A 50% (w/v) stock solution of MDI in acetone was

Effects of MDI exposure on human albumin

A rapid reaction (X + Y  Z) occurs when human albumin (X) is mixed with diisocyanate (Y), a class of chemicals known to cause asthma. The products of this reaction (Z) are antigenic to the human immune system but remain incompletely understood. We characterized the different reaction products between 4,4′-MDI and human albumin that result when a range of different starting reactant concentrations are present under the biologically relevant reaction conditions (see Materials and methods). Human

Discussion

This article has provided new insight into the antigenic changes that occur in the self protein, human albumin, when exposed to the occupational allergen MDI. Advances were made possible by combining several different analytical methods to characterize the range of MDI–albumin reaction products that form under different exposure conditions. Biologically relevant MDI–albumin reaction products produced in vitro were identified by serum IgG from MDI-exposed workers and further characterized

Acknowledgments

We thank the Yale Keck Center for its assistance with the HPLC and MS studies. We are especially thankful to Tom Abbott, Mary LoPresti, Kathy Stone, and Walt McMurray for their helpful guidance and data analysis. Funding was provided by the National Institute of Environmental Health and Safety through Grants R42-ES016728 and R41-ES018021 (to A.V.W.) and by the National Institute of Occupational Health and Safety (to C.A.R.).

References (37)

Cited by (37)

  • Immunochemical detection of the occupational allergen, methylene diphenyl diisocyanate (MDI), in situ

    2016, Journal of Immunological Methods
    Citation Excerpt :

    As shown (Fig. 1C), the MDI antisera contained IgG that bound to albumin conjugated with MDI, but not HDI or TDI, or unconjugated (mock exposed) albumin preparations. In contrast, control biotinylated anti-human-albumin goat IgG recognizes albumin bound by all 3 diisocyanates; however binding is reduced following diisocyanate conjugation, likely due to chemical modification of the native albumin structure as previously described (Wisnewski et al., 2008, 2010). We tested the ability of the MDI antisera to detect MDI in airway cells recovered by bronchoalveolar lavage (BAL).

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