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.

Characterizing Adduct Formation of Electrophilic Skin Allergens with Human Serum Albumin and Hemoglobin

“…Anyhow, respiratory allergens are also haptens that induce harmful health effects by activating the immune system in much the same way as contact allergens, with the main difference being that the symptom upon elicitation is asthma rather than eczema. Albumin and Hb adducts with diisocyanates have been studied both in vitro and in vivo , and it has been shown that the most abundant diisocyanate adducts are Lys-adducts from albumin 56 − 60 and N-terminal Val adducts of Hb. 60 − 63 Diisocyanate-adducts of albumin have been shown to be immunogenic 60 , 64 and are believed to be involved in the etiology of sensitization to the diisocyanates.…”

“…Albumin and Hb adducts with diisocyanates have been studied both in vitro and in vivo , and it has been shown that the most abundant diisocyanate adducts are Lys-adducts from albumin 56 − 60 and N-terminal Val adducts of Hb. 60 − 63 Diisocyanate-adducts of albumin have been shown to be immunogenic 60 , 64 and are believed to be involved in the etiology of sensitization to the diisocyanates. 65 , 66 Further, the levels of MDI-HSA adducts in MDI-exposed individuals have been shown to correlate with symptoms of allergic asthma and with the presence of diisocyanate-specific IgG antibodies.…”

“… 65 , 66 Further, the levels of MDI-HSA adducts in MDI-exposed individuals have been shown to correlate with symptoms of allergic asthma and with the presence of diisocyanate-specific IgG antibodies. 67 Diisocyanate-adducts of HB are not believed to be immunogenic 60 but are still considered useful for biomonitoring purposes. 62 , 63 The mean adduct levels of MDI-Lys adducts of HSA in construction workers exposed to MDI were found to be 102 fmol/mg HSA 57 (corresponding to 0.007 mol MDI-Lys/mol HSA), and the mean levels in MDI-exposed individuals suffering from MDI-induced allergic asthma were found to be 501 fmol/mg HSA 67 (corresponding to 0.03 mol MDI-Lys/mol HSA).…”

Serum Periostin Levels: A Potential Serologic Marker for Toluene Diisocyanate-Induced Occupational Asthma

“…Conjugation between TDI and HSA, cross-linking, and reactivity were demonstrated. 15 Among them, TDI-conjugated HSA at 40:1 was more reactive than the others in enzyme-linked immunosorbent assay (ELISA). Therefore, 40:1 TDI-HSA conjugate was used in this study.…”

Biomonitoring Human Albumin Adducts: The Past, the Present, and the Future

“…The experiments were performed with a high ratio (1:1, 5:1, 10:1, and 40:1) of isocyanates (MDI, HDI, TDI) to Alb. 228−230 The high concentrations used for the experiment greatly exceeded the levels of human exposure. In industrial workers, the median adduct level of MDI-Alb was 30 fmol/mg Alb (= 0.002 mol MDI-Lys/mol Alb).…”