title: Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin.
abstract: Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb-TDI antigenicity was approximately 30% that of HSA-TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity.
authors: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA; Department of Chemistry, Portland State University, Portland, OR 97207, USA.
authors: Morgen
authors: M
authors: Mhike
authors: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
authors: Justin M
authors: JM
authors: Hettick
authors: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
authors: Itai
authors: I
authors: Chipinda
authors: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
authors: Brandon F
authors: BF
authors: Law
authors: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
authors: Toni A
authors: TA
authors: Bledsoe
authors: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
authors: Angela R
authors: AR
authors: Lemons
authors: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
authors: Ajay P
authors: AP
authors: Nayak
authors: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
authors: Brett J
authors: BJ
authors: Green
authors: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
authors: Donald H
authors: DH
authors: Beezhold
authors: Department of Chemistry, Portland State University, Portland, OR 97207, USA.
authors: Reuben H
authors: RH
authors: Simoyi
authors: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.
authors: Paul D
authors: PD
authors: Siegel
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D006241:Haptens / Q000737:chemistry* / Q000276:immunology; D006454:Hemoglobins / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D017953:Isocyanates / Q000737:chemistry* / Q000276:immunology; D012709:Serum Albumin / Q000737:chemistry* / Q000276:immunology; D014051:Toluene 2,4-Diisocyanate / Q000737:chemistry* / Q000276:immunology
chemical_list: D003432:Cross-Linking Reagents; D006241:Haptens; D006454:Hemoglobins; D017953:Isocyanates; D012709:Serum Albumin; C015262:1,6-hexamethylene diisocyanate; D014051:Toluene 2,4-Diisocyanate
keywords: 1,6-Hexamethylene diisocyanate; 2,4-Toluene diisocyanate; Haptenation; Occupational asthma