Isocyanate vapor-induced antigenicity of human albumin

BACKGROUND: The bioreactivity of isocyanate, a leading cause of occupational asthma, has led to uncertainty regarding the chemical's antigenicity and mechanisms that elicit immunopathology. OBJECTIVE: To understand better the biologically relevant antigenic forms of hexamethylene diisocyanate (HDI), commonly used in the auto body industry. METHODS: Human albumin was exposed to HDI vapors through a novel approach designed to model the air-liquid interface of the human airway. Vapor HDI-exposed albumin was characterized by electrophoresis, chemical substitution analysis, mass spectrometry, and serology studies on auto body shop workers (N=203) and HDI asthmatics (N=11). RESULTS: HDI vapors caused significant changes in the shape and/or charge of human albumin, which differed from albumin exposed to liquid phase HDI, with lower isocyanate substitution ratios and distinct electrophoretic mobility. Specific sites of vapor HDI conjugation to albumin were identified at His(247) and Lys(414). Vapor HDI-exposed albumin was specifically recognized by the humoral arm of the human immune system, with a strong dependence on albumin as the carrier. Vapor HDI-exposed albumin-specific IgG titers were significantly associated with HDI exposure (P=.001), and specific IgE was detectable in 55% (6/11) of isocyanate asthmatics versus 1.5% (3/203) of exposed healthy workers. Parallel studies using HDI-exposed albumin conjugates produced by previously published methods showed less significant associations of HDI-specific IgG and IgE with exposure and disease, respectively. CONCLUSION: HDI-albumin conjugates produced by novel vapor phase exposure methods may be more physiologically relevant than those produced by previously published methods and of greater utility in characterizing the immune responses associated with HDI exposure and asthma.     

Evaluation of antibody binding to diisocyanate protein conjugates in a general population.

BACKGROUND: Specific IgG binding to diisocyanate-human serum albumin (HSA) has been proposed as an indicator of diisocyanate exposure. One residential study reported IgG binding to diisocyanate conjugates in 8% of residents living near a factory using toluene diisocyanate (TDI). Because comparable assays were not performed using individuals distant from such facilities, the significance of this finding is uncertain. OBJECTIVE: To determine the prevalence of diisocyanate specific antibodies in sera from individuals "not known to be exposed" to diisocyanates. METHODS: Serum samples from 139 anonymous donors without known diisocyanate exposure were assayed by means of enzyme-linked immunosorbent assay for IgG or IgE specific for TDI-HSA, diphenylmethane diisocyanate (MOI)-HSA, and hexamethylene diisocyanate (HDI)-HSA. Positive responses (optical density > or = 0.1 and > or = 3 SDs above the mean of 8 laboratory controls) were run 3 times. Competitive inhibition was performed for sera exhibiting binding of optical density of at least 0.2. RESULTS: We detected IgG reactive with HDI-HSA, diphenylmethane diisocyanate-HSA, and TDI-HSA in 18 (13%), 0, and 7 donors (5%), respectively. Inhibition (>50%) was demonstrated in 6 of 9 participants with elevated HDI-HSA levels and in 2 of 7 with elevated TDI-HSA levels. We detected IgE reactive with the same antigens in 3 donors (2%); however, none were confirmed to be positive using the biotin-streptavidin IgE assay. CONCLUSIONS: Specific and nonspecific IgG binding to HDI-HSA and TDI-HSA were detected in individuals without known exposure to isocyanates. These antibody measurements may not be reliable indicators of diisocyanate exposure in nonoccupational populations and should not be interpreted as surrogates of diisocyanate exposure in the absence of defined referent populations.     

IgG antibodies against polyisocyanates in car painters

A group of thirty car painters exposed to vapours and aerosols of paint containing prepolymer and monomer of hexametylene diisocyanate (HDI) was investigated. Specific antibodies against monomer HDI and prepolymerized HDI were analysed with RAST (IgE) and ELISA (IgG) assays after conjugation of the haptens with human serum albumin. There was no significant increase of serum IgG antibodies against HDI monomer, nor of specific IgE antibodies against HDI monomer or prepolymer. Specific IgG antibodies against prepolymerized HDI were significantly increased, as compared with non-exposed referents (medians 0.11 vs 0.03 absorbance (A)). Six car painters were found to have specific IgG antibodies of subclass 4 against HDI prepolymer, four also against HDI monomer. This shows an association between exposure and specific IgG antibodies. Thirteen subjects had suffered symptoms of rhinitis and/or conjunctivitis, and ten had symptoms from the bronchi (two asthma). There was no significant association between symptoms and levels of specific antibodies. Most of the symptoms were slight and unspecific, probably due to irritative effects of the exposure.     

A Study of Serum Antibody Activity in Workers with Occupational Exposure to Diphenylmethane Diisocyanate

The prevalence of sensitization was studied in a group of 76 foundry workers with occupational exposure to diphenylmethane diisocyanate (MDI). Ten workers had clinical evidence of asthma, 40 had non-asthmatic respiratory symptoms, and 26 were asymptomatic. Specific IgE antibodies to MDI were found in two workers (2.6%) and specific IgG antibodies, in five workers (6.6%). The prevalence of IgE and IgG antibodies was higher in the 10 subjects with asthma than in the non-asthmatic group. The prevalence of anti-paratolyl-monoisocyanate antibodies was not significantly different from that of anti-MDI antibodies, and both haptenic determinants displayed a high degree of cross-reactivity in the RAST inhibition test. The role of humoral immunological mechanisms in MDI-induced asthma is unclear in view of the rather low prevalence of these serum antibodies in this group of workers.     

The guinea pig model of diisocyanate sensitization. I. Immunologic studies

Two strains of guinea pigs were parenterally immunized with well-characterized diisocyanate-protein conjugates. Hapten-specific IgE antibodies were detected in the sera of English short-hair strain guinea pigs immunized with either toluene diisocyanate-human serum albumin (TDI-HSA) or hexamethylene diisocyanate-HSA (HDI-HSA) when these sera were analyzed by the 168 hr passive cutaneous anaphylaxis (PCA) technique followed by intravenous challenges with conjugates of respective ligands coupled to an unrelated carrier protein, transferrin. IgG1 antibodies and precipitating antibodies were demonstrated in Hartley strain guinea pigs immunized with TDI/HDI-HSA conjugates. The hapten specificity of these antibodies was proved by PCA inhibition experiments and antibody absorption experiments. In the precipitating antibody system, this was further confirmed by immunoelectrophoretic analysis. Cross-reactivity between HDI and TDI was not observed in the PCA experiments. However, apparent cross-reactivity in the double gel diffusion experiments was due to new antigenic determinants formed by isocyanates after conjugation with proteins. It was therefore apparent that immune responses of guinea pigs immunized with protein conjugates of bifunctional isocyanates were heterogeneous and involved multiple specificities for hapten, carrier protein, and new antigenic determinants. It was postulated that the complex nature of the immune response generated by diisocyanate compounds in the guinea pig may also serve as a more appropriate model of isocyanate-induced human sensitivity reactions, which are known to involve diverse immunologic and nonimmunologic mechanisms.     

Biophysical determinants of toluene diisocyanate antigenicity associated with exposure and asthma

BACKGROUND: Toluene diisocyanate (TDI), a widely used aromatic diisocyanate with the potential to cause asthma, reacts with albumin in the airway fluid, which acts as a carrier protein for chemical presentation to the immune system. Structural elucidation of TDI-albumin conjugates is crucial to understanding the human immune response to TDI exposure. OBJECTIVE: Investigate the dependence of TDI's antigenicity on the biophysics of exposure and its association with TDI asthma. METHODS: Toluene diisocyanate-albumin conjugates were generated by exposing albumin to TDI in liquid or vapor phase (liquid or vapor TDI-albumin, respectively). Conjugates were characterized by native gel electrophoresis and matrix-assisted laser desorption/ionization-mass spectrometry, and used as antigens in ELISA assays for serum specific-IgE and IgG. RESULTS: The physical phase of TDI (vapor vs liquid) affects the formation of TDI-albumin conjugates, with measurable differences in the amount of TDI per albumin molecule, migration in native gels, matrix-assisted laser desorption/ionization-mass spectrometry mass/charge spectra, and antigenicity. Vapor TDI-albumin conjugates were recognized by IgE from 44% of subjects with TDI asthma, whereas liquid TDI-albumin conjugates are recognized by IgE from only 17% of these patients. A significant (P < .05) association between TDI exposure and vapor TDI-albumin specific serum IgG was also observed. CONCLUSION: Biophysics of TDI exposure substantially affects formation of TDI-albumin conjugates recognized by the immune system in association with exposure and asthma. CLINICAL IMPLICATIONS: The data suggest that serology may help identify TDI asthmatics and exposed workers if the appropriate form of TDI is used as the antigenic basis for analysis.     

Experiences of specific IgE in asthma due to diisocyanates

The specific IgE antibodies were studied with the Phadebas® RAST technique in 35 patients with asthma due to diisocyanates. Two had been sensitized to toluene diisocyanate (TDI), 17 to methylene diisocyanate (MDI) and 16 to hexamethylene diisocyanate (HDI). In each case the diagnosis was confirmed with a bronchial provocation test (BPT). The asthmatic reaction was immediate in 17 cases, of which three had also a late reaction (dual). Eighteen patients reacted only with a late reaction. Seven (20%) had specific IgE to diisocyanates. All RAST-positive patients had an immediate asthmatic reaction. None of the late reactors and referents had positive RASTs. RAST inhibition tests with 94–100% inhibition confirmed the specificity of the method. There was cross-reactivity between different diisocyanates, however. The patients with positive RAST to diisocyanates had a higher level of total IgE than the RAST negative group and the referents.     

Diisocyanate asthma and gene-environment interactions with IL4RA, CD-14, and IL-13 genes.

BACKGROUND: Diisocyanate asthma (DA) affects 2% to 10% of exposed workers, yet the pathogenetic mechanisms underlying this disorder remain ill defined. OBJECTIVE: To determine if specific single nucleotide polymorphisms (SNPs) of interleukin 4 receptor alpha (IL4RA), IL-13, and CD14 promoter genes are associated with DA. METHODS: Sixty-two workers with DA confirmed by specific inhalation challenge (SIC) and 75 diisocyanate-exposed, SIC-negative workers were analyzed for SNPs associated with IL4RA, IL-13, and CD14 promoter genes. RESULTS: No associations were found with individual SNPs and DA. When stratified according to specific diisocyanate exposure, a significant association was found between IL4RA (I50V) II and DA among individuals exposed to hexamethylene diisocyanate (HDI) (odds ratio [OR], 3.29; 95% confidence interval [CI], 1.33-8.14; P = .01) only. Similarly, the IL4RA (I50V) II and IL-13 (R110Q) RR combination was significantly associated with DA in HDI-exposed workers (OR, 4.13; 95% CI, 1.35-12.68; P = .01), as was the IL4RA (I50V) II and CD14 (C159T) CT genotype combination (OR, 5.2; 95% CI, 1.82-14.88; P = .002) and the triple genotype combination IL4RA (I50V) II, IL-13 (R110Q) RR, and CD14 (C159T) CT (OR, 6.4; 95% CI, 1.57-26.12; P = .01). CONCLUSIONS: Gene-environmental interactions may contribute to the pathogenesis of DA, and gene-gene interactions may modulate this relationship.     

Immunologic specificity of isocyanate-induced IgE antibodies in serum from 10 sensitized workers

A procedure for the preparation of RAST disks used to assay isocyanate-specific IgE antibodies was developed. The outcome of the RAST was found to be strongly dependent on how the isocyanate test antigen was synthesized. Specific IgE from selected workers with isocyanate asthma reacted optimally to conjugates with less than or equal to 10 isocyanate molecules bound per molecule of human serum albumin. Further haptenization resulted in decreased specific binding and increased nonspecific binding because of high levels of total IgE. The hapten and carrier specificity of isocyanate-induced IgE antibodies were studied by direct RAST and RAST inhibition. The existence of new antigenic determinants related to both the isocyanate hapten and the carrier could be demonstrated. It was important to use a test antigen prepared from the same isocyanate as that to which the worker had been exposed, since the cross-reactivity between different isocyanate haptens was partial and varied from one patient to another. It was confirmed that isocyanate-specific IgE antibodies can be demonstrated only in a subgroup of workers with isocyanate-related bronchial asthma.     

Occupational asthma and specific IgE to a diazonium salt intermediate used in the polymer industry

This study describes sensitization to a diazonium salt intermediate, diazonium tetrafluoroborate (DTFB), produced during the manufacture of a fluorine polymer precursor. Most of the workers exposed to DTFB powder complained of respiratory and mucosal irritation. However, some of these individuals had symptoms typical of occupational asthma. Clinical and immunologic studies, including bronchial provocation testing and specific IgE measurements, were performed on two individuals with asthmatic symptoms associated with exposure to DTFB. These studies confirmed a diagnosis of occupational asthma. In an investigation of 43 other exposed workers in two separate factories handling the diazonium compound, specific IgE antibodies to DTFB-human serum albumin conjugate were found in 20% of individuals. There was a good correlation between specific IgE and exposure-related respiratory symptoms, which suggests an IgE-mediated pathogenesis. DTFB-human serum albumin conjugates were characterized by electrophoretic techniques, and it was found that conjugates prepared at pH 10 elicited optimum IgE binding in RAST. In vitro cross-linking of albumin by DTFB was demonstrated but had no effect on RAST binding. Our findings on the effect of pH and polymerization on IgE binding support proposed mechanisms for in vivo conjugation and provide information on the antigenic determinants important in IgE recognition of hapten-carrier protein conjugates.     

Clinical and immunologic studies among egg-processing workers with occupational asthma

Twenty-five workers in an egg-processing factory were evaluated for respiratory sensitization to inhaled egg proteins by a physician evaluation, serial peak expiratory flow rate (PEFR) measurements for a 1-week period, and immunologic tests. Immunologic studies included skin prick tests, serum-specific IgE (RAST), and specific IgG (ELISA) to solutions prepared from commercial food allergens: factory-powdered egg white and yolk products and purified egg white fractions, including ovalbumin, ovomucoid, lysozyme, and conalbumin. Six workers had significant daily PEFR lability (greater than 20%) of whom five had associated cutaneous reactivity to at least one egg allergen. A diagnosis of "definite asthma" was established in five workers suspected by the physician of having asthma. These five workers exhibited significant decrements in daily PEFR that were accompanied by bronchial symptoms. Occupational asthma was diagnosed by the physician in four of the five latter workers. Definite asthma was significantly associated with both cutaneous reactivity to egg allergens (p less than 0.01) and RAST binding (p less than 0.01). Of eight workers with cutaneous reactivity to at least one egg reagent, four workers (50%) were positive to only purified egg white fractions. The highest levels of RAST binding were detected in four workers, and the best binding activity was to ovomucoid and ovalbumin fractions. Elevated specific IgG responses were significantly higher in egg-factory workers to whole egg (p less than 0.005), lysozyme (p less than 0.002), and conalbumin (p less than 0.002) allergens compared to responses of nonexposed control subjects. However, no differences in specific IgG were detected between symptomatic and asymptomatic workers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical and immunological evaluation of isocyanate-exposed workers.

Isocyanates are the most significant cause of occupational asthma in our country. To evaluate the prevalence of work-related respiratory symptoms and immunologic sensitization to it, we performed a questionnaire survey, allergy skin test, radioallergosorbent test (RAST) to toluene diisocyanate (TDI)-human serum albumin (HSA) conjugate and methacholine bronchial challenge test on 23 isocyanate-exposed employees and 9 unexposed controls working in a zipper factory. Six employees (26.1%) complained of work-related respiratory symptoms and three symptomatic workers showed significant bronchoconstrictions on TDI-bronchoprovocation test. Three (13%) asymptomatic workers had high specific IgE antibodies to TDI-HSA and none of the TDI-sensitive asthmatic workers had specific IgE antibody. One of the TDI-sensitive asthmatic workers showed a negative result on the initial methacholine bronchial challenge test, but bronchial hyperresponsiveness developed after the TDI challenge. It was suggested that TDI-sensitive asthma was noted in three (13%) of 23 exposed workers and that asymptomatic workers could have high specific IgE antibody. Measurement of the changes in bronchial hyperresponsiveness after the TDI challenge could be helpful to diagnose TDI-sensitive asthma.     

Are allergen-specific IgG mainly IgG anti-IgE autoantibodies?

A large proportion of specific IgE occurred in immune complexes with anti-IgE autoantibodies in sera from nonhyposensitized allergic patients. These autoantibodies were misinterpreted as 'specific IgG' in different immunoassays such as dot immunoassays and the radioallergosorbent test (RAST), leading to overestimation of specific IgG. Purified immune complexes contained even more IgG than IgE antibodies. Heating of the complexes liberated specific IgE, producing an upwards RAST class shift. Thus anti-IgE autoantibodies are hiding the specific IgE, which is thereby underestimated. It is not known whether the hiding anti-IgE autoantibodies are also effectively neutralizing circulating or cell-bound IgE and might represent the actual blocking antibody.     

Detection of specific IgE in isocyanate and phthalic anhydride exposed workers: comparison of RAST RIA, Immuno CAP System FEIA, and Magic Lite SQ

Sera from 94 workers occupationally exposed to isocyanate were tested by RAST RIA, Immuno CAP FEIA, and Magic Lite SQ (ML) against the allergens HDI, MDI, TDI, and phthalic anhydride. Twenty sera showed increased levels of diisocyanate-specific IgE antibodies, and five sera were phthalic anhydride positive. High total IgE titer was not correlated with positive specific IgE titer ( r = 0.47), showing that nonspecific IgE binding was low. The results of ML and CAP correlated well ( r = 0.91), but tended to be slightly higher than the results obtained with the RAST isotope test. CAP and RAST data also correlated significantly ( r = 0.90), but the correlation between ML and RAST data was lower ( r = 0.82). The results of the new in vitro tests, CAP and ML, showed good reproducibility (5% CV for CAP and 8% CV for ML). In summary, the CAP and ML methods were found to be appropriate for routine diagnosis of specific IgE antibodies against the allergens HDI, MDI, TDI, and phthalic anhydride.     

An optimized assay of specific IgE antibodies to reactive dyes and studies of immunologic responses in exposed workers

Methods of assaying reactive dye-specific IgE antibodies were investigated with a RAST. Sera from three patients, occupationally exposed to a reactive dye, Remazol black B (Chemical Abstract registry number 17095-24-8), were used. Directly dyed disks, that is, disks without any carrier protein, resulted in poor and unreliable measures of specific IgE. In contrast, optimized preparation of conjugates between the dye and human serum albumin resulted in efficient binding of specific IgE. The patients' RAST results were strongly positive, whereas sera from 36 exposed workers but without symptoms and sera from unexposed subjects with high levels of total IgE were negative. The hapten and carrier specificity of the IgE antibodies was studied by direct RAST and RAST inhibition. In one patient, the antibodies were principally hapten specific, whereas another patient was found to have antibodies with a high degree of specificity to the carrier. The third patient's antibodies were intermediate between the other two patients' antibodies in this respect, suggesting that antibody specificity is dependent not only on the nature of the hapten but also on individual immune response factors. The study demonstrates that it is important to use an optimized preparation of dye-protein conjugates to elicit reliable results and a high degree of specific IgE binding in the RAST.     

Immunological specificity of chloramine-T-induced IgE antibodies in serum from a sensitized worker

A procedure for the preparation of chloramine-T (CT) conjugates used to assay IgE antibodies was developed using response surface methodology and serum from a subject occupationally exposed to the substance. The conjugates, synthesized by reacting CT with human serum albumin (HSA) and other protein carriers, were used as antigens in a radio-allergosorbent test (RAST). Human serum albumin was found to be a suitable carrier, although other protein carriers also gave specific IgE-binding of a similar extent. The CT-HSA conjugates used in the RAST were characterized by high performance liquid chromatography, electrophoresis, immunodiffusion and ammonium sulphate precipitation. However, no strong correlation was seen between the ability of the conjugates to bind IgE and their physical or immuno-chemical properties. The hapten and carrier specificity of CT-induced IgE antibodies in the subject's serum were studied by direct RAST and RAST inhibition. No existence of new antigenic determinants related to the carrier could be demonstrated. Although HSA as a carrier was altered immunochemically by CT, the IgE antibodies were found to be specific to hapten only. Chloramine-T-specific IgG antibodies could not be demonstrated in the subject's serum.     

Immunologic tests of specific antibodies to organic acid anhydrides

The outcome of immunologic tests of antibodies directed against hapten conjugates of organic acid anhydrides and human serum albumin (HSA) has been studied in workers exposed to phthalic anhydride (PA), methyltetrahydrophthalic anhydride (MTHPA), hexahydrophthalic anhydride (HHPA), and methylhexahydrophthalic anhydride (MHHPA). HSA conjugates of PA, MTHPA, HHPA, MHHPA, and maleic anhydride (MA) have been prepared and used in the tests. The hapten densities (HD) of the conjugates were varied by different molar ratios of hapten and macromolecule in the preparative procedure. Skin prick reactions to MTHPA-HSA increased with rising HD over the range 6-13 mol/mol. The achieved HD was tested by spectrometric and gas chromatographic methods. In RAST of IgE antibodies MTHPA-HSA with HD six and 25 showed significantly lower bindings than conjugates with intermediate HD. There was a good correlation between skin prick tests and RAST. Of 234 workers tested [MTHPA (n = 145), and HHPA (n = 89)], 45 had a skin prick reaction greater than or equal to 50% of the histamine reaction (1 mg/ml). All but two of these were RAST positive (RAST value greater than 0.3%; 0.3% upper range in 147 controls; MTHPA, n = 63; HHPA, n = 84). Nine RAST positive workers had no obvious skin prick reaction. However, their RAST values were low (less than 0.8%). In exposed workers, the ELISA value of specific IgG antibodies to MTHPA-HSA showed optimal values when tested with the HD 13 conjugate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomarkers predicting isocyanate-induced asthma

THREE DIISOCYANATES CAN CAUSE OCCUPATIONAL ASTHMA (OA): toluene diisocyanate (TDI), 4,4 diphenylmethane diisocyanate (MDI), and 1,6-hexamethylene diisocyanate (HDI). We analyzed potential biomarkers of isocyanate-induced OA, based on investigated immunologic, genetic, neurogenic, and protein markers, because there is no serological testing method. The prevalence of serum IgG to cytokeratin (CK)18 and CK19 in TDI-OA was significantly higher than in controls, although the prevalence of these antibodies was too low for them to be used as biomarkers. Another candidate biomarker was serum IgG to tissue transglutaminase (tTG), because the prevalence of serum specific IgG to tTG was significantly higher in patients with TDI-OA than in controls. The human leukocyte antigen (HLA) DRB1*1501-DQB1*0602-DPB1*0501 haplotype may be used as a genetic marker for TDI-OA in Koreans via enhanced specific IgE sensitization in exposed subjects. The genetic polymorphisms of catenin alpha 3, alpha-T catenin (CTNNA3) were significantly associated with TDI-OA. Additionally, examining the neurokinin 2 receptor (NK2R) 7853G>A and 11424 G>A polymorphisms, the NK2R 7853GG genotype had higher serum vascular endothelial growth factor (VEGF) levels than the GA or AA genotypes among Korean workers exposed to TDI. To identify new serologic markers using a proteomic approach, differentially expressed proteins between subjects with MDI-OA and asymptomatic exposed controls in a Korean population showed that the optimal serum cutoff levels were 69.8 ng/mL for ferritin and 2.5 μg/mL for transferrin. When these two parameters were combined, the sensitivity was 71.4% and the specificity was 85.7%. The serum cytokine matrix metalloproteinase-9 (MMP-9) level is a useful biomarker for identifying cases of TDI-OA among exposed workers. Despite these possible biomarkers, more effort should be focused on developing early diagnostic biomarkers using a comprehensive approach based on the pathogenic mechanisms of isocyanate-induced OA.     

Diverse profiles of specific IgE response to toluene diisocyanate (TDI)-human serum albumin conjugate in TDI-induced asthma patients

The prevalence studies on specific IgE to toluene diisocyanate (TDI)-human serum albumin (HSA) conjugate in TDI-induced asthma have shown variable results. In this study, we attempted to compare specific IgE bindings to TDI-HSA conjugate and its specificity using 3 different conjugates. Sera were collected from 20 TDI-induced asthma and 10 controls. Specific IgE were measured by ELISA using three TDI-HSA conjugates; two from Carnegie Mellon (CM; 98 and 99 CM conjugates) and one from Ajou University. To evaluate specificity and cross-reactivity, ELISA inhibition tests were applied. Positive and negative predictive values between Ajou conjugate and 98 CM conjugate were 75% and 100%. Those between Ajou and 99 CM were 100% and 93.8%. One patient showed an isolated positive response to the Ajou with negative responses to the other two conjugates. ELISA inhibition test using this patient's serum revealed the significant inhibitions by the Ajou and minimal inhibitions by the others. On the other hand, another patient showed an isolated positive response to 99 CM with negative responses to the others, and ELISA inhibition test showed significant inhibition by 99 CM with minimal inhibitions by the others. These results suggest that specific IgE bindings to a new antigenic determinant of TDI-HSA conjugate can be heterogeneous and differ from one individual to another.