Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin
| Autor: | Angela R. Lemons, Justin M. Hettick, Toni A. Bledsoe, Donald H. Beezhold, Morgen Mhike, Brett J. Green, Paul D. Siegel, Reuben H. Simoyi, Brandon F. Law, Itai Chipinda, Ajay P. Nayak |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Antigenicity medicine.drug_class Immunology Serum albumin Enzyme-Linked Immunosorbent Assay Monoclonal antibody Article 03 medical and health sciences chemistry.chemical_compound Hemoglobins 0302 clinical medicine medicine Immunology and Allergy Humans Serum Albumin Chromatography biology medicine.diagnostic_test Human serum albumin 030210 environmental & occupational health 030104 developmental biology Cross-Linking Reagents chemistry Immunoassay biology.protein Hexamethylene diisocyanate Toluene 2 4-Diisocyanate Hapten Haptens Keyhole limpet hemocyanin medicine.drug Isocyanates |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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