title: The influence of diisocyanate antigen preparation methodology on monoclonal and serum antibody recognition.
abstract: Exposure to diisocyanates (dNCOs), such as methylene diphenyl diisocyanate (MDI) can cause occupational asthma (OA). Currently, lab tests for dNCO specific IgE are specific, but not sensitive, which limits their utility in diagnosing dNCO asthma. This may be due to variable preparation and poor characterization of the standard antigens utilized in these assays. The aim of this study was to produce and characterize a panel of antigens prepared using three different commonly employed methods and one novel method. The conjugates were examined for recognition by anti-MDI monoclonal antibodies (mAbs) in varying enzyme linked immunosorbant assay (ELISA) formats, extent of crosslinking, total amount of MDI, the sites of MDI conjugation, relative shape/charge, and reactivity with human serum with antibodies from sensitized, exposed workers. Results indicate that while there are minimal differences in the total amount of MDI conjugated, the extent of crosslinking, and the conjugation sites, there are significant differences in the recognition of differently prepared conjugates by mAbs. Native and denaturing polyacrylamide gel electrophoresis demonstrate differences in the mobility of different conjugates, indicative of structural changes that are likely important for antigenicity. While mAbs exhibited differential binding to different conjugates, polyclonal serum antibodies from MDI exposed workers exhibited equivalent binding to different conjugates by ELISA. While differences in the recognition of the different conjugates exist by mAb detection, differences in antigenicity could not be detected using human serum from MDI-sensitized individuals. Thus, although dNCO conjugate preparation can, depending on the immunoassay platform, influence binding of specific antibody clones, serologic detection of the dNCO-exposure-induced polyclonal antibody response may be less sensitive to these differences.
authors.affiliation: a Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention , Morgantown West Virginia.
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Hagerman
authors.affiliation: a Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention , Morgantown West Virginia.
authors.forename: Brandon F
authors.initials: BF
authors.lastname: Law
authors.affiliation: a Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention , Morgantown West Virginia.
authors.forename: Toni A
authors.initials: TA
authors.lastname: Bledsoe
authors.affiliation: a Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention , Morgantown West Virginia.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Hettick
authors.affiliation: b Biostatistics and Epidemiology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention , Morgantown , West Virginia.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Kashon
authors.affiliation: a Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention , Morgantown West Virginia.
authors.forename: Angela R
authors.initials: AR
authors.lastname: Lemons
authors.affiliation: c Department of Internal Medicine , Yale School of Medicine , New Haven , Connecticut.
authors.forename: Adam V
authors.initials: AV
authors.lastname: Wisnewski
authors.affiliation: a Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention , Morgantown West Virginia.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Siegel
mesh_terms: D000941:Antigens / Q000032:analysis* / Q000737:chemistry; D059366:Asthma, Occupational / Q000139:chemically induced / Q000175:diagnosis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes; D017953:Isocyanates / Q000276:immunology*; D016273:Occupational Exposure; D001559:Benzhydryl Compounds
chemical_list: D000941:Antigens; D000939:Epitopes; D017953:Isocyanates; C005969:4,4'-diphenylmethane diisocyanate; D001559:Benzhydryl Compounds
keywords: Diisocyanate; ELISA; MDI; enzyme-linked immunosorbant assay; methylene diphenyl diisocyanate