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

Evaluation of antibody binding to diisocyanate protein conjugates in a general population David I. Bernstein, MD*; M. Gerald Ott, PhD†; Michael Woolhiser, PhD‡; Zana Lummus, PhD*; and Cynthia Graham, PhD§

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 ⱖ0.1 and ⱖ3 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. Ann Allergy Asthma Immunol. 2006;97:357–364.

INTRODUCTION Serum specific IgE and IgG antibodies to diisocyanates have been widely investigated as diagnostic markers of occupational asthma in diisocyanate-exposed workers.1–12 Initially, radioimmunoassay methods were used,3,9,13,14 but in recent years, enzyme immunoassay methods, including enzymelinked immunosorbent assays (ELISAs),11,12,15 enzyme allergosorbent tests, and CAP-FEIA (Pharmacia, Uppsala, Sweden),16 have become more prominent. In 3 studies5,10,12 of workers undergoing specific inhalation challenge, elevated specific IgE evaluated by means of ELISA identified specific inhalation challenge–positive workers with 14% to 31% sensitivity and 89% to 97% specificity. The corresponding sensitivity and specificity estimates for specific IgG were 46% to 72% and 74% to 92%, respectively. Based on such data, it has generally been accepted that diisocyanate specific antibodies lack the sensitivity to distinguish workers with occupational * Division of Immunology and Allergy, University of Cincinnati, Cincinnati, Ohio. † Corporate Medical Department, BASF Corp, Rockaway, New Jersey ‡ Toxicology & Environmental Research & Consulting, The Dow Chemical Co, Midland, Michigan. § Product Environmental Health and Safety Management, Huntsman LLC, The Woodlands, Texas. Received for publication January 16, 2006. Accepted for publication in revised form March 27, 2006.

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asthma from diisocyanate-exposed workers who are asymptomatic. Specific IgG antibodies against diisocyanate– human serum albumin (HSA) conjugates have been proposed as useful markers of previous occupational exposure to diisocyanates.17–21 In a study of 455 isocyanate-exposed and 157 unexposed individuals, Selden and coworkers17 reported specific IgG antibody in 5% to 10% of polyurethane workers and spray painters and in 0% of control employees. Similarly, in a prospective study19 of 150 Canadian spray painters, hexamethylene diisocyanate (HDI)–HSA specific IgG antibodies were detected by means of ELISA in 13% of employees without work-related respiratory symptoms. In an occupational setting where ambient air exposures to diphenylmethane diisocyanate (MDI) were generally well controlled, only 2 of 243 employees were found to have specific IgG responses to MDI-HSA. These individuals were currently assigned to work tasks involving the direct use of MDI-resin mixtures to mend imperfections in the final product.22 One of the latter employees experienced work-related immediateonset urticaria and facial angioedema but no respiratory symptoms. In a workplace study of 174 employees, Skarping and colleagues20 found that workers positive for MDI-HSA specific IgG (n ⫽ 35) had higher concentrations of urinary and plasma MDI metabolites. The prevalence of work-related symptoms was only marginally different between specific

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IgG–positive and specific IgG–negative employees (31% vs 22%). Binding of antibodies to diisocyanate conjugates has also been evaluated as exposure markers in residential populations living close to polyurethane foam production facilities. In a Finnish residential population living near a foam production site, specific IgE antibodies to toluene diisocyanate (TDI), HDI, or MDI were detected by means of radioallergosorbent testing in 1 of 62 residents identified by questionnaire as having asthma.23 However, the single positive response was observed in a resident who reported previous occupational exposure to diisocyanates. In a US study,24 serum samples from 113 residents living near a flexible foam facility were analyzed by means of ELISA for specific IgE and IgG antibodies against TDI-HSA, MDI-HSA, and HDI-HSA conjugates. Ten residents (9%), including 1 with occupational diisocyanate exposure and 2 with possible exposure to diisocyanate products, had positive responses to at least 1 of the 3 conjugates. These investigators concluded that exposure to TDI in ambient air may have been responsible for the positive antibody responses in some residents. The findings of this and similar studies are difficult to interpret in the absence of appropriate comparison populations. Comparisons across studies are further complicated by a lack of standardization in the methods used to assay for diisocyanate specific antibodies.21 The present study was undertaken to determine the prevalence of diisocyanate specific antibodies in serum samples from a population of blood donors “not known to be exposed” to diisocyanates using the same immunoassay method as in the previous US residential study.24 METHODS Participants and Study Design Eligible participants were anonymous blood donors recruited through Biological Specialty Corp (Colmar, PA). All the participants provided written informed consent before entry into the study. Because the identity of participants was anonymous, the University of Cincinnati institutional review board exempted investigators from obtaining additional informed consent for the purpose of performing diisocyanate specific antibody assays. Anonymous donors had blood samples collected at medical centers in Pennsylvania and Florida and completed a brief 13-item questionnaire. Questionnaire items captured the demographic profile of the study group (age and sex), characterization of possible work- and hobby-related exposure to products containing diisocyanates, smoking history, allergy and asthma status, respiratory symptoms, and recent respiratory tract infections. Questionnaire responses were used to identify individuals with no known isocyanate exposure and those with possible work- or hobby-related exposure to diisocyanates. Thirty milliliters of blood was collected for serum analysis from each participant. An aliquot of each serum sample was shipped frozen to the University of Cincinnati for analysis. A sample size of 150 was selected based

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on a determination that this sample size would provide reasonably narrow confidence intervals for prevalence estimates, expecting that 5% or fewer donors would test positive for at least 1 diisocyanate-HSA conjugate. Conjugate Preparation and Characterization The HDI-HSA, MDI-HSA, and TDI-HSA conjugates were prepared following the methods of Tse and Pesce.25 The conjugates were sterilized by means of 0.2-␮m membrane filtration, and protein concentration was determined using the BioRad procedure. For determination of the hapten:protein molar ratio, conjugates were exhaustively dialyzed vs deionized water, and mass spectrometry was performed at the University of Cincinnati Mass Spectrometry Laboratory. Diisocyanate-HSA conjugates were compared with mocktreated HSA for determination of the increased mass attributable to diisocyanate substitution and the diisocyanate-HSA molar ratio. The hapten-protein molar ratios of the 3 conjugates used in this study were determined to be 22.8 for HDI:HSA, 2.6 for MDI:HSA, and 19.3 for TDI:HSA. ELISA Procedures Diisocyanate specific IgE and IgG antibodies were assayed in triplicate by means of indirect ELISA detection of the immunocomplexes with a labeled immunoglobulin,26 as previously described.12 High-binding microplates (Costar 9018; Corning Inc, Corning, NY) were coated with 0.1 mL of 10 ␮g/mL of diisocyanate-HSA or mock-conjugated HSA carrier protein in 0.15M sodium chloride by incubating for 2 hours at 37°C and overnight at room temperature. Plates were washed with phosphate-buffered saline (PBS) (0.01M sodium phosphate, 0.14M sodium chloride, 0.02% [wt/vol] sodium azide, 0.05% [wt/vol] Tween 20, pH 7.4) and blocked for 1 hour with blocking buffer (IgG assay: 5% bovine serum albumin in PBS; IgE assay: 1% ovalbumin in PBS). A positive reference serum sample, with anti– diisocyanate IgE and IgG antibodies, was obtained from a diisocyanate-exposed worker with confirmed occupational asthma. Reference control serum samples were obtained from 8 individuals with no known exposure to diisocyanates and were preselected for negativity (optical density [OD] ⬍0.1) compared with the known positive control (OD ⬎0.6). Sera from these individuals have been repeatedly used in this laboratory as referents for defining a positive assay result and controlling for plate-to-plate variability in the assay. Specific IgE and IgG were assayed at 1:10 and 1:100 dilutions of test sera. Plates were incubated at room temperature for 1 hour, with 0.1 mL of test or control serum per well. The IgG immunocomplexes were detected using goat anti– human IgG (␥-chain and species specific, alkaline phosphatase labeled, 1:20,000 dilution, 0.1 mL/well) (product No. 3187; Sigma-Aldrich Corp, St Louis, MO). The IgE immunocomplexes were detected using unlabeled goat anti– human IgE (Fc specific, affinity purified IgG, 100 ng/well), followed by rabbit anti– goat IgG (␥-chain specific, alkaline phosphatase labeled, 50 ng/well) (KPL, Gaithersburg, MD).

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The IgE antibodies were also assayed using a biotinstreptavidin ELISA procedure.27 To achieve optimal test sensitivity, sample and reagent volumes were increased to 0.2 mL/well, all incubations were performed at 37°C, and Tris buffer was substituted for phosphate buffer. Antigen-coated plates were washed with Tris-buffered saline (0.1M Tris hydrochloride, 0.15M sodium chloride, 0.02% sodium azide, 0.05% Tween 20, pH 7.4) and blocked for 1 hour with blocking buffer (Tris-buffered saline containing 2% bovine serum albumin). Test and control sera were added and incubated for 1 hour, then IgE immunocomplexes were detected with biotinylated goat anti– human IgE (Fc specific, 100 ng/well) (KPL), followed by streptavidin-alkaline phosphatase (100 ng/well) (KPL). For all the ELISAs, the substrate consisted of 1 mg/mL of p-nitrophenyl phosphate in 10% (wt/vol) diethanolamine and 0.5-mmol/L magnesium chloride, pH 9.8. Positive and negative control serum samples were included on each plate, and plates were read for specific IgE and IgG when the positive reference serum (1:10 dilution) triplicates reached a mean OD405 nm of 0.6. For specific IgE and IgG, a test was considered positive if the mean OD405 nm of test serum (1:10 dilution) triplicates was at least 0.1 and also at least 3 SDs above the mean of 8 negative reference serum samples for 3 separate ELISA runs. In addition, the tested serum must have yielded a negative response for antibody to mock-conjugated HSA. Inhibition Studies Competitive inhibition assays28 were performed for all the sera exhibiting positive binding to any diisocyanate-HSA antigen with an OD of at least 0.2. Aliquots of test sera, diluted 1:5, were mixed with equal volumes of blocking buffer (antibody control); 1, 10, 100, or 1,000 ␮g/mL of diisocyanate-HSA conjugate; or 1, 10, 100, or 1,000 ␮g/mL of mock-conjugated HSA. The aliquots were then incubated overnight on a rotary shaker at room temperature. The inhibition mixtures were then added to antigen-coated plates, and IgG- or IgE-specific ELISA tests were performed according to the protocols described previously herein. Percentage of inhibition was calculated as follows: 100 ⫻ 关共Mean OD of Uninhibited Serum ⫺ Mean OD of Inhibited Serum)/Mean OD of Uninhibited Serum] Test results showing at least 50% inhibition using a nydiisocyanate antigen concentration tested, without concomitant inhibition by HSA (⬍50% inhibition at any HSA concentration and consistently less than inhibition by antigen), were considered indicative of serum specific antibody reactions. Statistical Analysis Data management and analysis were performed using a software program (SAS Version 8; SAS Institute, Cary, NC). Outcome variables of interest included (1) the OD readings on the first run for each conjugate, antibody class, and dilution tested; (2) the ELISA outcome (positive or negative) based on confirmation on 3 independent runs; and (3) the

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result of the inhibition test (when performed). Potential explanatory variables included age, sex, current cigarette smoking, allergy and asthma symptoms, occupation, and classification with respect to potential diisocyanate exposure. Analysis of positive and negative ELISA responses was based on the Proc FREQ procedure, and OD readings were examined by means of rank statistics methods (Proc NPAR1WAY). RESULTS One hundred fifty donors provided serum samples and completed the aforementioned questionnaire. A review of the responses identified 4 persons who had worked in facilities using diisocyanates and 7 who had reported contact with diisocyanate-containing products through other work or home activities. Results of these 11 persons considered to have had previous diisocyanate exposure are summarized and discussed separately. Occupation, cigarette smoking history, and allergy and asthma status of the 139 donors not known to be exposed are summarized in Table 1. Fifty-six (40%) were women, and 83 (60%) were men. Donors ranged in age from 19 to 60 years, with the mean age of women and men being 32.4 and 32.8 years, respectively. By occupation, 46% of the men performed construction, maintenance, or transportation work, whereas 94% of the women were employed in services (including homemaking), sales, and office work or did not report an occupation. Two thirds of the men and women reported current smoking. Allergy symptoms were reported by 12% of Table 1. Selected Characteristics of 139 Donors With No Known Exposure to Diisocyanates Donors, No. (%) Characteristic

Age, y 18–24 25–34 35–44 45–60 Occupation Construction/maintenance Food services Sales/office Services* Transport Miscellaneous Not working/students Cigarette smoking history Current smoker Ex-smoker Nonsmoker Not reported Allergy symptoms Asthma symptoms

Women (n ⴝ 56)

Men (n ⴝ 83)

17 (30) 18 (32) 12 (21) 9 (16)

23 (28) 27 (33) 21 (25) 12 (14)

2 (4) 5 (9) 9 (16) 20 (36) 1 (2) 9 (16) 10 (18)

20 (24) 13 (16) 5 (6) 4 (5) 18 (22) 7 (8) 16 (19)

37 (66) 3 (5) 16 (29) 0 10 (18) 5 (9)

56 (67) 10 (12) 16 (19) 1 (1) 7 (8) 3 (4)

* Includes homemakers.

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the respondents and asthma symptoms by 6%, with women more frequently reporting these findings than men. The 9 donors who reported experiencing shortness of breath, chest tightness, cough, or wheezing were all current cigarette smokers. As mentioned previously herein, 11 donors were identified with possible previous exposure to diisocyanates. Their mean age was 35.7 years. Eight were men, and 91% reported current cigarette smoking. Only 1 of these 11 donors reported symptoms of allergies, and none reported symptoms of asthma. Immunoassay Results The numbers and percentages of positive immunoassay results among the 139 donors are given in Table 2 by antibody class and conjugate. Positive samples were defined as having OD values of at least 0.10 that exceeded the mean ⫹3 SDs of the 8 negative referent samples on 3 separate ELISA runs. The 29 positive test results were detected in 24 different donors. The ELISA inhibition tests were performed on 19 samples that had demonstrated a level of binding of OD of 0.2 or greater. Overall, 3 (2%) of 139 nonexposed donors had positive specific IgE readings by means of the indirect sandwich assay for any of the diisocyanate conjugates, and 22 donors (16%) had positive specific IgG readings. Inhibition (ⱖ50% reduction without concomitant HSA inhibition) was demonstrated in 1 of 3 positive IgE samples and in 9 of 16 positive IgG samples. IgG-specific inhibition was observed in 6 (67%) of 9 participants tested with elevated HDI-HSA binding and in 2 (29%) of 7 individuals tested with elevated TDI-HSA binding. The biotin-streptavidin IgE assay detected no IgE binding in any of the 3 donors exhibiting significant IgE

Table 2. Positive Binding by Antibody Class and Conjugate in 139 Donors With No Known or Likely Diisocyanate Exposure Antibody class and conjugate IgE class‡ HDI conjugate MDI conjugate TDI conjugate IgG class HDI conjugate MDI conjugate TDI conjugate

No.

% (95% CI)

Inhibition test, No. positive/ No. tested†

1 1 2

0.7 (0.0–3.9) 0.7 (0.0–3.9) 1.4 (1.7–5.1)

0/1 0/1 1/1

18 0 7

12.9 (7.9–19.7) 0 (0.0–2.6) 5.0 (2.1–10.1)

6/9 0/0 2/7

Positive results*

Abbreviations: CI, confidence interval; HDI, hexamethylene diisocyanate; MDI, diphenylmethane diisocyanate; TDI, toluene diisocyanate. * Positive readings on 3 enzyme-linked immunosorbent assays (3 separate runs) at 1:10 dilution of test sera. † Inhibition assays were run on only 19 of 29 samples exhibiting positive binding to any diisocyanate– human serum albumin antigen; the optical density readings were too low (⬍0.2) to assay 10 “positive” samples. ‡ Data obtained from enzyme-linked immunosorbent assay indirect specific IgE assay (see the “Methods” section).

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binding via the indirect sandwich assay, which used a second detection antibody (rabbit anti– goat IgG); this includes the single sample that demonstrated specific IgE inhibition. Of the 11 donors with possible occupational or nonoccupational diisocyanate exposure, 2 (18%) had positive specific IgE or IgG responses. In 1 donor, inhibition tests did not reveal specificity for IgE binding with HDI-HSA and TDIHSA or for IgG binding with TDI-HSA, but IgG for HDIHSA was inhibited. This donor reported neither allergy nor asthma symptoms. The second donor was positive for IgG HDI-HSA, but inhibition tests could not be performed because the OD values were less than 0.2 in 2 of 3 runs. Frequency Distributions of Antibody Binding The frequency distributions of OD readings for IgG responses by conjugate are plotted in Figures 1 and 2 for the 139 nonexposed donors. Individual results are shaded in gray if positive on the first run but not on a subsequent run and in black if confirmed positive based on 2 additional runs. Each individual donor value was adjusted for plate-to-plate differences by subtracting the mean OD of the 8 negative referent samples included on each plate for control purposes. The adjusted OD readings varied from ⫺0.09 to 0.30. The modal value of each of the 4 histograms was 0.00. None of the individual readings approached the value of the positive control sample included on each plate. The spread of OD values for specific IgG was least for the MDI-HSA conjugate, with none of the adjusted OD readings exceeding 0.10 (Fig 2). The higher-substituted conjugates (HDI-HSA and TDIHSA) were less peaked in distribution and had larger interquartile ranges (Fig 1) compared with MDI-HSA and HSA alone (Fig 2). Intra-assay and interassay variability of OD readings for HDI-HSA and TDI-HSA conjugates was estimated based on samples run in triplicate on the same plate and replicated 3 times on different plates. These samples had OD readings averaging 0.17 for HDI-HSA and 0.27 for TDI-HSA conjugates. The mean intra-assay coefficients of variation (CVs) were 7% and 11% for HDI and TDI conjugates, respectively, and the corresponding interassay CVs were 39% for both conjugates averaged across all samples tested. The interassay standard deviations observed with these samples were similar to that observed for the positive laboratory control subject; however, the CVs for the HDIand TDI-positive controls were 12% and 7%, respectively, due to the higher mean OD values. Antibody Responses by Donor Characteristics Of the 139 donors with no known occupational or hobby contact with diisocyanates, immunoassay responses (specific IgE and IgG for each of the 3 diisocyanate conjugates and HSA alone) were also examined in relation to 6 host characteristics: age, sex, cigarette smoking, allergy symptoms, symptoms of asthma, and occupational category. Rank correlation analyses were performed based on adjusted OD readings, and contingency table analyses were performed categorizing antibody responses as positive or negative where a

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Figure 1. Frequency distribution of first-run optical density (OD) values (donor ⫺ negative control mean) for IgG toluene diisocyanate (TDI)– human serum albumin (HSA) and hexamethylene diisocyanate (HDI)–HSA at 1:10 dilution for 139 donor samples. Neg indicates negative; pos, positive.

Figure 2. Frequency distribution of first-run optical density (OD) values (donor ⫺ negative control mean) for IgG human serum albumin (HSA) and diphenylmethane diisocyanate (MDI)–HSA at 1:10 dilution for 139 donor samples. Neg indicates negative; pos, positive.

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positive test result was confirmed on 3 separate runs. The only consistent pattern was that OD readings for specific IgG tended to be decreased for all the conjugates, including HSA alone, among current cigarette smokers and persons reporting allergy symptoms (data not shown). The corresponding contingency table analyses revealed no statistically significant differences with respect to any of the 6 factors when the outcome was a positive test result confirmed on 3 separate runs. The same analysis was also run for the 11 donors reporting possible diisocyanate contact. No statistically significant differences were seen in either the rank analyses or the analyses based on the percentage of positive test results compared with the 139 nonexposed donors. Effect of Serum Dilution on Assay Outcome During the first run, samples from all the donors were analyzed at 2 different serum dilutions, 1:10 and 1:100. These results were run on the same microplates, thus limiting potential sources of experimental error. The percentage of positive test results was decreased considerably at the 1:100 dilution compared with the 1:10 dilution across conjugates and antibody classes. For HDI and TDI conjugates, decreases were observed for IgE-HDI (from 7.9% to 0.7%), IgG-HDI (from 23.7% to 2.2%), IgE-TDI (9.4% to 1.4%), and IgG-TDI (from 10.8% to 2.9%). DISCUSSION In this study, we demonstrated the presence of IgG antibodies reactive with HDI-HSA and TDI-HSA in individuals with no known exposure to diisocyanates. These findings were confirmed and reproduced on 3 separate ELISA runs. In most positive samples, IgG binding was detectable at a serum titer of 1:10 only and yielded low OD readings compared with the positive control serum. The prevalence estimates among individuals without known exposure to diisocyanates are at variance with some previous estimates for nonexposed populations. For example, Selden and colleagues17 reported zero prevalence in 157 nonexposed workers. In their study, the minimum requirement for a positive finding was an absorbance value of at least 0.5 OD using a 1:20 serum dilution level and confirmation by means of ELISA inhibition. Several other investigators20,29,30 who included unexposed controls in their studies defined the cutoff point for a positive response as the highest value in the control group. Similar to us, some investigators defined a positive response as 2 to 3 SDs above the mean of unexposed control subjects.31 In the present study, a positive response was defined as a mean OD405 nm of test serum triplicates of at least 0.1 and also 3 SDs or more above the mean results for 8 negative laboratory controls on 3 separate ELISA runs. Thus, differences in study outcomes could be attributed to varying criteria for defining a positive result. In measuring IgE reactive with HDI-HSA, TDI-HSA, and MDI-HSA, a few positive IgE-binding responses were detected using an indirect sandwich assay. However, only 1 of these samples demonstrated specificity via ELISA inhibition,

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and none could be confirmed using the biotin-streptavidin IgE assay. The positive IgE results seen using the sandwich assay may be explained by cross-reactivity of the second detection antibody (rabbit anti-goat) recognizing human IgG bound to the plate. Although the use of multiple layers of species-specific detection antibodies may enhance assay sensitivity, this could occur at the expense of reduced specificity. Certain commercial immunoglobulin reagents are cross-reactive across species, and this could lead to the detection of false-positive IgE reactions when secondary detection antibodies react with human IgG bound to the plate. This phenomenon was recently demonstrated with the indirect specific IgE immunoassay (data not shown) and might be responsible for some false-positive IgE test results. Although many investigations of anti– diisocyanate IgE antibodies via ELISA have used some variation of a double sandwich indirect method,5,12,19 one such study24 was conducted in a residential setting near a flexible foam production facility using the same indirect ELISA methods as we have for detection of IgE and IgG antibodies to HDI-HSA, MDIHSA, and TDI-HSA conjugates. Nine residents exhibited elevated IgG levels to one or more diisocyanate conjugates, but one of these residents had reported previous occupational exposure to diisocyanates, and another indicated possible exposure to diisocyanate-containing varnishes. Excluding these individuals with possible exposure (as done in this study), 7 residents (6%) had positive responses with the TDI-HSA conjugate in the absence of identifiable exposure to diisocyanates. From these data, the authors surmised that some of the positive antibody responses could have resulted from ambient exposure to TDI near the facility.24 However, the specificity of the IgG responses was not confirmed by means of inhibition assay, and a demographically matched population of residents living distant to the factory was not evaluated. This would have enabled determination of a background frequency of diisocyanate specific antibodies in their population based on the specific method used in the assay. In the present study, we found a 5% background prevalence of positive IgG binding reactions with TDI-HSA in a population-based sample without known exposure to diisocyanates. This approximates the 6% prevalence of IgG binding to the TDI-HSA conjugate reported in the previous residential study.24 In addition, only 1 sample in this residential study24 showed positive IgG binding to HDI-HSA, whereas in the present study, 13% of the serum samples tested showed positive binding to HDI-HSA, and 7 were positive by inhibition assay. The reasons for the discrepant results with respect to the HDI conjugates are unknown. We performed ELISA inhibition on 19 samples using homologous conjugates to evaluate the possibility of nonspecific binding. Significant inhibition of IgG binding was demonstrated in 6 (67%) of 9 HDI-HSA and 2 (29%) of 7 TDI-HSA assays, suggesting the possibility of nonspecific binding in half of the positive IgG samples, even after 3 ELISA runs. Whereas conformational change, denaturation, and possible acquisition of new antigenic sites might occur

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after passive adsorption of protein antigens onto polystyrene,32–35 little is known regarding conjugate hapten structure, especially as it relates to the availability of antigenic sites in solution vs those bound to a microtiter plate. Thus, further work is necessary to understand this method for hapten antigens. The origin of the specific IgG measured in the present study among persons without known diisocyanate exposure is uncertain. The process of classifying serum samples as positive, and comparing results across studies, is complicated by a lack of standardization in the methods used to assay for diisocyanate specific antibodies. The criteria used for classification of positive samples are variable and somewhat arbitrary. Interlaboratory studies with ELISA protocols have observed decreased precision and agreement in testing of serum samples with known low levels of antibody.36 Although the number of participants reporting potential diisocyanate exposure was small (n ⫽ 11) in this study, results for these individuals were not notably different from those for the nonexposed donors. It is possible (although unproved) that immune responses to diisocyanate-HSA conjugates could be elicited by previous exposure to isocyanates or isothiocyanates from environmental sources. For example, vegetables, including radishes, are rich sources of isothiocyanates, and 1 case of concurrent bronchial reactivity to TDI and radishes has been described in the literature.37 Polyurethane polymers are incorporated into a variety of industrial, medical, and consumer products, and some adhesives and other consumer products may contain isocyanates. The detection of residual isocyanate moieties in some polyurethane products has only been reported using solvent extraction techniques,38 – 40 and vapor concentrations of TDI cannot be detected in fresh polyurethane foam 24 hours after production.41 In the present study, the lack of correlation between specific IgG responses and donor characteristics, such as age, sex, allergy status, and occupation, does not point to any particular occupation or environmental source as an explanatory factor for the positive responses. The use of highly substituted conjugates (HDI-HSA and TDI-HSA but not MDI-HSA) could have also contributed to the number of positive specific IgG responses. No positive responses were detected to MDI-HSA, the least-substituted conjugate. Increased IgE or IgG binding with conjugates having higher diisocyanate:HSA ratios has been demonstrated in several laboratories and seems to be nonspecific, possibly related to conformational changes in the protein structure itself.7,21,42,43 In applying preset criteria for defining a positive test result and confirming positive test results by replication across runs and by inhibition assay, we demonstrated a positive background rate of specific IgG for several diisocyanate conjugates. We did not identify particular host factors (eg, sex, age, smoking history, symptom history, or occupation) that were predictive of positive antibody responses. These data, showing positive responses in the absence of identifiable diisocyanate exposure, suggest that these antibody measurements may

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not be reliable indicators of diisocyanate exposure in nonoccupational populations and should not be interpreted as surrogates of diisocyanate exposure in the absence of a comparison with defined referent populations. REFERENCES 1. Baur X, Dewair M, Fruhmann G. Detection of immunologically sensitized isocyanate workers by RAST and intracutaneous skin tests. J Allergy Clin Immunol. 1984;73:610 – 618. 2. Pezzini A, Riviera A, Pagiaro P, et al. Specific IgE antibodies in twenty-eight workers with diisocyanate-induced bronchial asthma. Clin Allergy. 1984;14:453– 461. 3. Tse KS, Johnson A, Chan H, et al. A study of serum antibody activity in diphenylmethane diisocyanate exposed workers. Allergy. 1985;46:314 –320. 4. Keskinen H, Tupasela O, Tiikkainen U, et al. Experiences of specific IgE in asthma due to diisocyanates. Clin Allergy. 1988; 18:597– 604. 5. Cartier A, Grammer L, Malo J-L, et al. Specific serum antibodies against isocyanates: association with occupational asthma. J Allergy Clin Immunol. 1989;84:507–514. 6. Cvitanovic S, Zekan L, Marusic M. Occurrence and specificity of IgE antibodies to isocyanates in occupational exposed workers. Int Arch Occup Environ Health 1989;61:483– 486. 7. Wass U, Belin L. Immunologic specificity of isocyanateinduced IgE antibodies in serum from 10 sensitized workers. J Allergy Clin Immunol. 1989;83:126 –135. 8. Karol MH, Tollerud DJ, Campbell TP, et al. Predictive value of airways hyperresponsiveness and circulating IgE for identifying types of responses to toluene diisocyanate inhalation challenge. Am J Respir Crit Care Med. 1994;149:611– 615. 9. Tee RD, Cullinan P, Welch J, et al. Specific IgE to isocyanates: a useful diagnostic role in occupational asthma. J Allergy Clin Immunol. 1998;101:709 –715. 10. Park H-S, Kin H-Y, Nahm D-H, Son J-J, Kim Y-Y. Specific IgG, but not specific IgE, antibodies to toluene diisocyanate– human serum albumin conjugate are associated with toluene diisocyanate bronchoprovocation test results. J Allergy Clin Immunol. 1999;104:847– 851. 11. Aul DJ, Bhaumik A, Kennedy AL, et al. Specific IgG response to monomeric and polymeric diphenylmethane diisocyanate conjugates in subjects with respiratory reactions to isocyanates. J Allergy Clin Immunol. 1999;103:749 –755. 12. Bernstein DI, Cartier A, Cote J, et al. Diisocyanate antigen–simulated monocyte chemoattractant protein-1 synthesis has a greater test efficiency than specific antibodies for identification of diisocyanate asthma. Am J Respir Crit Care Med. 2002;166:445– 450. 13. Patterson R, Harris KE, Zeiss CR. Antibodies against toluene diisocyanate protein conjugates: three methods of measurement. J Allergy Clin Immunol. 1983;72:676 – 680. 14. Gallagher JS, Tse CS, Brooks SM, et al. Diverse profiles of immunoreactivity in toluene diisocyanate (TDI) asthma. J Occup Med. 1981;23:610 – 616. 15. Park HS, Lee SK, Kim HY, et al. Specific immunoglobulin E and immunoglobulin G antibodies to toluene diisocyanate– human serum albumin conjugate: useful markers for predicting longterm prognosis in toluene diisocyanate–induced asthma. Clin Exp Allergy. 2002;32:551–555. 16. Baur X, Chen Z, Flagge A, et al. EAST and CAP specificity for the evaluation of IgE and IgG antibodies to diisocyanate-HSA

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Requests for reprints should be addressed to: David Bernstein, MD Division of Immunology University of Cincinnati 231 Albert Sabin Way ML 563 Cincinnati, OH 45267-0563 E-mail: [email protected]

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