title: Development of sandwich ELISAs for the detection of aromatic diisocyanate adducts.
abstract: Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals commonly used in the manufacturing industry. Occupational exposures to dNCOs have been shown to elicit allergic sensitization and occupational asthma. Among the most commonly used dNCOs in industry are the aromatic dNCOs, toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI). This study aimed to develop enzyme linked immunosorbent assays (ELISA) utilizing aromatic dNCO-specific monoclonal antibodies (mAbs) for the detection of aromatic dNCO adducts. Two sandwich ELISAs were developed. The first sandwich ELISA utilized mAb 60G2 along with an anti-human serum albumin (HSA) polyclonal antibody. This assay detected MDI-, 2,4- and 2,6-TDI-HSA adducts with limits of detection (LOD) of 2.67, <0.10, and 1.70 ng/mL, respectively. When spiked into human serum, the LOD of this ELISA increased to 34.37, 7.64 and 24.06 ng/mL, respectively. The second ELISA utilized mAbs 62G5 and 60G2 for capture and detection. This assay was capable of detecting 2,4- and 2,6-TDI-HSA adducts with LODs of <4.90 and 26.92 ng/mL, respectively, and when spiked in human serum, <4.90 and 95.93 ng/mL, respectively. This 62G5-60G2 sandwich assay was also able to detect dNCO adducted transferrin, hemoglobin, keratin and actin, but with less sensitivity than dNCO-HSA. The results of this study demonstrate potential application of these ELISAs in the identification and characterization of aromatic dNCO adducts as well as in biomonitoring occupational and environmental dNCO exposures.
authors.affiliation: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, 1095 Willowdale Road, Morgantown, WV 26505, USA. Electronic address: wrw0@cdc.gov.
authors.forename: Angela R
authors.initials: AR
authors.lastname: Lemons
authors.forename: Toni A
authors.initials: TA
authors.lastname: Bledsoe
authors.forename: Paul D
authors.initials: PD
authors.lastname: Siegel
authors.forename: Donald H
authors.initials: DH
authors.lastname: Beezhold
authors.forename: Brett J
authors.initials: BJ
authors.lastname: Green
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D017953:Isocyanates / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D012709:Serum Albumin / Q000276:immunology; D014051:Toluene 2,4-Diisocyanate / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D001559:Benzhydryl Compounds
chemical_list: D000911:Antibodies, Monoclonal; D017953:Isocyanates; D012709:Serum Albumin; D014051:Toluene 2,4-Diisocyanate; C005969:4,4'-diphenylmethane diisocyanate; D001559:Benzhydryl Compounds
keywords: Diisocyanate; ELISA; HDI; HSA; Immunoassay; MDI; Monoclonal antibody; Occupational asthma; TDI; dNCO; diisocyanate; enzyme linked immunosorbent assay; hexamethylene diisocyanate; human serum albumin.; mAb; methylene diphenyl diisocyanate; monoclonal antibody; toluene diisocyanate