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Chem. Res. Toxicol. 1988, 1, 288-293
Diisocyanate Antigens That Detect Specific Antibodies in Exposed Workers and Guinea Pigs Ruzhi Jint and Meryl H. Karol* Jilin Provincial Institute of Industrial Health and Occupational Disease, Changchun, Jilin Province, People's Republic of China, and Department of Industrial Environmental Health Sciences, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received M a y 19,1988
Evaluation of the immunologic contribution to the pathogenesis of isocyanate lung disease necessitates preparation of isocyanate-protein conjugates to detect anti-isocyanate antibodies. The preparation and characterization of the conjugates were described in the preceding paper in this issue. Sera were obtained from two guinea pigs immunized with 4,4'-diisocyanatodiphenylmethane (MDI) and from three workers with occupational exposure to MDI. By use of Western blot analysis, guinea pig IgG antibodies were best detected by the monomeric component of MDI-guinea pig serum albumin. ELISA additionally indicated that conjugates which contained a high density of hapten detected greater amounts of antibody than did conjugates containing low amounts of hapten. The same procedures were then used to assess the amount of MDI-specific IgG and IgE antibody in sera from symptomatic workers. Effective MDI-HSA antigens were those that were monomeric and had high haptenic content. Highly substituted conjugates of monoisocyanates (phenyl isocyanate and p-tolyl isocyanate) with serum albumins were also effective in detecting antibodies to MDI. These results indicate the importance of the composition of isocyanate conjugate antigens in detecting antibodies to diisocyanates and suggest that standards be developed for preparation and characterization of these diagnostic serologic reagents.
Introduction The ability of industrial diisocyanates to cause pulmonary hypersensitivity is well documented (1,2).Symptoms include immediate- and late-onset asthmatic reactions (3), hypersensitivity pneumonitis (4),cough, and dyspnea (5). Although there is increased recognition of the relationship between the exposure levels of the chemicals and the development of sensitivity (6-9),the mechanismb) underlying these responses is (are) still uncertain. Several investigators have reported frequent identification of specific antibodies in sera from exposed and sensitized individuals (6,10, 11), whereas others have detected antibodies in only a small proportion of symptomatic workers (12).Many reasons have been suggested for this discrepancy, the most frequent being differences in the preparation of the antigen used in the particular immunoassay (1,2, 13,14). We undertook a study to determine the influence of preparation conditions on the nature of the products formed from reaction of the industrial diisocyanate MDI with serum albumin. The latter is the protein most frequently used in preparation of hapten conjugates as diagnostic reagents. The reaction products were found to possess various degrees of haptenic content as well as intraand intermolecular cross-linked species dependent upon the conditions of the reaction. The questions we posed were the following: Did the conjugates differ in ability to detect antibodies to MDI? What were the characteristics of hapten-conjugates that *To whom correspondence should be addressed at the University of Pittsburgh. + Jilin Provincial Institute of Industrial Health and Occupational Disease.
best detected diisocyanate-specificantibodies of various classes in animals and in industrial workers? This study attempted to answer both questions.
Materials and Methods Animals. Male, English short hair guinea pigs (Hilltop Lab Animals, Inc., Scottdale, PA) weighing approximately 400 g were used for production of antibodies. Immunizationof Animals. Two guinea pigs were immunized by intradermal injection with 50 pL of a 10% solution of MDI in acetone into each of two shaved sites in the dorsal region. Twenty-five days later they received a second set of injections of 50 pL of 6% MDI in acetone into each of two naive dorsal sites. Blood was drawn from the nailbed prior to immunization and by cardiac withdrawal on days 33 and 36. The two postimmunization samples from each animal were combined and stored at -20 "C until use. Diisocyanate-Protein Conjugates. Conjugation of monoand diisocyanates with human (or guinea pig) serum albumin, (HSA, GSA) was performed as described in the previous paper (15). Briefly, 4,4'-diisocyanatodiphenylmethane(MDI, Mondur M, Mobay Chemical Corp.) was recrystallized from n-hexane. It was dissolved in acetone and added dropwise to a rapidly stirred solution of 0.5% serum albumin in buffer. The following parameters were varied to produce conjugates with varying degrees of haptenic substitution and inter- and intramolecular crow-linking bonds: initial molar ratio of isocyanate to protein, pH, temperature, and time of reaction. Reactions were terminated by addition of excess monoethanolamine. Conjugates were isolated by dialysis and then lyophilization. The amount of hapten bound per mole of protein was determined spectroscopically from the ultraviolet absorption of the conjugate. Human Subjects. Sera were obtained from three workers with industrial exposure to MDI. Each had symptoms suggestive of sensitization. Two (subjects A and B) had chest tightness and severe shortness of breath while a t work. The third (subject C) 0 1988 American Chemical Society
Detection of Isocyanate Antibodies had worked in the plant for 2 years without experiencing symptoms. Following exposure to an MDI spill, he developed respiratory tract irritation and chest tightness. Serum samples were drawn 2 months after the accidental exposure. Control subjects were individuals either who had never worked with MDI or who had no history of accidental MDI exposure. Serologic Assays. (1) Total Serum IgE. The amount of IgE in serum samples was determined by using a commercial IgE kit (Ventrex). (2) RAST (Radioallergosorbent Test). RAST was performed by coupling MDIIS-HSA to cyanogen bromide activated paper disks according to a standard procedure (6). For assay, 50 pL of serum was incubated with a washed disk at ambient temperature for 16 h. Disks were thoroughly washed, and then 9 - l a beled anti-human IgE (Ventrex Reagent) was added. After an additional 16 h at ambient temperature, disks were washed and then counted in a y counter (Packard). Titers were expressed as the percent of added radioactivity that remained bound to the disks. (3) Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate sera for the presence of antibodies to MDI. The procedure was adapted from that of Engvall and Perlmann (16). Ninety-six-wellpoly(viny1 chloride) microtiter plates (Flow Laboratories, McLean, VA) were coated by addition to each well of 50 pL of antigen conjugate (5 g / m L in 0.1 M carbonate buffer, pH 9.6). Plates were placed at 37 "C for 3 h and then stored at 4 "C. Immediately before use, wells were washed three times with saline containing 0.05% Tween 20. Fifty microliters of serum was added and serially diluted with PBS-Tween containing 0.5% bovine serum albumin (BSA, Sigma Chemical Co.). Plates were incubated at 37 "C for 2 h and then washed. Fifty microliters of a 1:800 dilution of rabbit anti-guinea pig IgG (H and L specific, Miles Scientific) conjugated to alkaline phosphatase was added and incubation allowed for 16 h at ambient temperature. Plates were washed and enzyme was assayed by using 1mg/mL disodium p-nitrophenyl phosphate (Sigma) in 1 M diethanolamine containing 0.5 mM MgC12. After incubation at 37 "C for 30 min, reactions were stopped by addition of 50 pL of 1 N NaOH. Absorbance was determined at 410 nm by using a Dynatech plate reader. Antibody titer was defined as the highest serum dilution that gave an absorbance at least twice that of the control sera and greater than 0.030 after subtraction of background absorbance. (4) ELISA Inhibition. Assays were performed by incorporating increasing quantities of antigens as inhibitors into the microtiter plates prior to addition of sera. Thereafter, the ELISA procedure was followed. Inhibition assays were performed in triplicate. The percent inhibition was calculated as absorbance with inhibitor % inhibition = 100 absorbance without inhibitor (5) Western Blot Assay. The procedure of Towbin et al. (17) was followed. Samples were applied to 4-22.5% gradient SDSPAGE gels, and electrophoresiswas performed at 10 "C by using 600 V, 30 W, and 50 mA. Fractions were transferred electrophoretically to nitrocellulose paper (0.45" pore size, Bio-Rad Corp.) overnight by using 50 mA, with cooling at 4-10 "C. The gel was tested for completenessof transfer by staining a peripheral strip with Coomassie Blue R250. Sheets were blocked by using 3% BSA in PBS, (12 mM sodium potassium phosphate, 0.17 M NaC1, 3.4 mM KCl, pH 7.6) for 1 h at ambient temperature, followed by additionally blocking with 1% BSA-1 % normal rabbit serum in PBS,. For assay, strips were incubated with human serum (1:50) or guinea pig serum (1:lOO) for 2 h at ambient temperature. Bound antibody was detected by using horseradish peroxidase conjugated rabbit anti-guinea pig IgG (1:200, H + L, ICN Immunobiologicals) or goat anti-human IgG (1:200, H + L, ICN). Following incubation for 2 h at room temperature, nitrocellulose sheets were washed and enzyme was detected by using a solution containing 0.06% 4-chloro-1-naphthol,10% methanol, 0.02 M PBS,, and 0.1% hydrogen peroxide. Human IgE antibody was detected by addition of 1251-labeled goat anti-human IgE (Ventrex)and incubation of strips at ambient temperature for 12 h. The strips were washed thoroughly and then cut into 0.55 X 0.60 cm2pieces and placed into glass tubes for counting (Packard y scintillation counter). For control purposes, serum from nonexposed individuals but containing the
Chem. Res. Toxicol., Vol. 1, No. 5, 1988 289 Table I. MDI-Specific Antibodies in Guinea Pigs following Injection with 10% MDI antibodv titeP animal no. HSA MD1,-HSA MDI,,-HSA MD1,-HSA 5 50.0 >50.0 >50.0 ~~
ELISA plates were coated with 5 pg/mL MD1,-HSA or 5 pg of MD1,-HSA. *Assays were performed at 1:20 serum dilution. GP, guinea pig. CAssayswere performed at 1:160 serum dilution. GP, guinea pig. same amount of total IgE as experimental serum was used.
Results Production of MDI-Specific Antibodies in Guinea Pigs. Blood was drawn from two guinea pigs following injection with MDI a n d evaluated for specific antibodies by using as t h e test antigen MD14,-GSA (43 mol of MDI/mol of GSA) in a n ELISA assay. Titers of 1:8192 and 1:4096 were detected for animals 5 and 6, respectively. T h e specificity of t h e antibodies for MDI was confirmed by hapten inhibition assay. Test inhibitors included GSA conjugates containing diverse aromatic mono- a n d diisocyanates, all of which h a d 39-43 mol of hapten/mol of protein as determined spectrophotometrically. Results of t h e inhibition assays are shown in Figure 1. Only MD14,-GSA was able t o inhibit t h e reaction of sera with MDI,,-GSA-coated microtiter plates. T h e absence of inhibition by GSA or any of t h e other aromatic isocyanate conjugates indicated high affinity of t h e antisera for t h e MDI hapten. D i f f e r e n t i a l Reaction of Antisera with MDI Conjugates. Antibodies were evaluated for reaction with several MDI-HSA conjugates containing a range of haptenic substitution (MDI,,-HSA, MDI12-HSA, a n d MD14-HSA). Values for one animal are shown in Figure 2. T h e serum demonstrated highest titers, i.e., 1:2560 and 1:1280, when assayed by using MDI,-HSA and MDII2HSA antigens, respectively. When assayed b y using t h e MD14-HSA, it typically demonstrated a titer of only 1:320. No antibody reactivity was detected with HSA. Results of ELISA assays were identical for both animals as summarized in Table I. Inhibition assays were performed t o compare t h e specificities of antibodies reactive with MD1,-HSA with those binding to MD14-HSA. Assays were performed by using microtiter plates coated with MDI,-HSA, and t h e n with plates coated with MD14-HSA. Results of inhibition assays are illustrated in Figure 3 a n d given in Table 11. With both animals, antibodies reactive with MD1,-HSA were inhibited effectively by MDI,,-HSA with quantities of 0.25 a n d 0.07 pg required t o achieve 50% inhibition of reaction for animal 5 and 6, respectively. MDI12-HSA was
290 Chem. Res. Toxicol., Vol. 1, No. 5, 1988
Jin and Karol
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Figure 1. ELISA inhibition assays of antibodies raised in guinea pigs to MDI. ELISA was performed with microtiter plates coated with MDI,-GSA. Increasing amounts of inhibitors were added to the coated plates just prior to addition of antisera. Amounts per well are shown. Solid squares indicate reactions of serum (150) from animal 5; hatched squares indicate reactions of serum (150) from animal 6. Symbols a t lower right indicate absence of inhibition with TD14,-GSA (A), TII-GSA (@), GSA ( O ) , and PhB-GSA (0). 0.4
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Figure 3. ELISA inhibition assay of serum from an individual animal using microtiter wells coated with (A) M D I r H S A or (B) MD14-HSA. Inhibitors were added to microtiter wells just prior to addition of serum. From the regression lines, the amounts required to achieve 50% inhibition of reaction are listed in Table 11.
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Figure 2. ELISA to detect antibodies reactive with MDI-HSA conjugates. Wells of microtiter plates were coated with MD14HSA, MDIrHSA, MDIsHSA, or HSA at 5 pg/mL. Serum from guinea pig 5 (120) was added to the first well, and serial 2-fold dilutions were made. Absorbance was assessed a t 410 nm, and titers were calculated after subtraction of values in wells containing no serum.
also effective, whereas much greater quantities of MDb-HSA were needed to achieve the same result. HSA had no inhibitory ability. Antibodies reactive with MD14-HSA were best inhibited by MDI,,-HSA, followed by MDI,-HSA then M D I s H S A (Figure 3B). No inhibition was afforded by HSA. The latter results indicated that, although the antigen which was bound to the wells contained few haptenic groups, the groups were essential for recognition by antibody. The ELISA inhibition results were consistent, with the interpretation that a population of antibodies was produced as a result of immunization of animals with MDI. Most of the antibodies showed specificity for a multihaptenic conjugate; a smaller population of antibodies recognized a less densely substituted conjugate.
Figure 4. Western blot assay to detect reactivity of guinea pig serum with isocyanate antigen conjugates. Lane A depicts the SDS-PAGE profile of MDIa-GSA stained with Coomassie blue. Western blot assay lane B, MDL-GSA; lane C, pT4,-GSA; lane D, Phm-GSA; lane E, TD142-GSA; F, GSA.
Reaction of Guinea Pig Sera with Polymeric Species. By use of the Western blot procedure, antisera from the guinea pigs were examined for preferential reactivity with the various polymeric species comprising the diisocyanate-conjugate antigens. Typical findings are
Chem. Res. Toxicol., Vol. 1, No. 5, 1988 291
Detection of Isocyanate Antibodies Table 111. Antibody to MDI in Workers Evaluated by ELISA and RAST Assays Using MDI-HSA Antigens total IgE RAST (7% serum (IU/mL)= ELISA~ binding) worker A 5 2000 2.0 worker B 11 4000 1.8 worker C 120
