| Development of Immunoassays for Biomonitoring of Hexamethylene Diisocyanate Exposure Ranulfo Lemus,1 Lina Lukinskeine,1 Mark E. Bier,2 Adam V. Wisnewski,3 Carrie A. Redlich,3 and Meryl H. Karol1 1Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 2Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA; 3Department of Internal Medicine, Yale University, New Haven, Connecticut, USA
Abstract
Hexamethylene diisocyanate (HDI) is used widely to manufacture polyurethanes for paints and coatings. It is an irritant and a chemical asthmagen. The U.S. Occupational Safety and Health Administration time-weighted average permissible exposure limit is 5 ppb and the ceiling limit is 20 ppb. We sought to develop a sensitive and specific immuno-bioassay to supplement workplace air monitoring and detect recent HDI exposure. For this, we produced rabbit antiserum to HDI-adducted keyhole limpet hemocyanin (HDI-KLH) . The specificity of the antiserum was demonstrated by its reaction with a variety of HDI-conjugated proteins and the absence of reactions with conjugates of other diisocyanates, namely toluene diisocyanate and diphenyl methylene diisocyanate. Four immunoassays were developed and compared for their ability to detect decreasing quantities of HDI-adducted human serum albumin (HSA) containing 2 mol HDI adduct per mol HSA (HDI2-HSA) as determined by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry. The sensitivities of some of the assays are within the range (0.82-45 nM) of current analytic methods. A Western analysis procedure has a sensitivity of 600 nM HDI adduct on HSA. ELISA inhibition assay, in which microtiter plates are coated with the HDI2-HSA antigen, has a sensitivity of 300 nM HDI adduct. An immunoblot assay has a sensitivity of 9 nM HDI adduct. The most sensitive bioassay (1.8 nM HDI adduct) is a three-antibody sandwich ELISA in which wells of microtiter plates are coated with the IgG fraction of the anti-HDI-KLH antisera. Compared with analytic methods for HDI biomonitoring, the immunoassays are faster and less costly and accommodate numerous samples simultaneously. The assays have the potential to affect industrial biomonitoring programs significantly. Key words: biomonitoring, HDI, immunoassay, occupational asthma. Environ Health Perspect 109:1103-1108 (2001) . [Online 11 October 2001] http://ehpnet1.niehs.nih.gov/docs/2001/109p1103-1108lemus/ abstract.html Address correspondence to M.H. Karol, 130 DeSoto Street, Pittsburgh, PA 15261 USA. Telephone: (412) 624-3155. Fax: (412) 624-3040. E-mail: mhk+@pitt.edu This study was supported by ES 05651 and OH 3457. Received 5 February 2001 ; accepted 10 April 2001.
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