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Specific IgE antibodies to reactive dye-albumin conjugates
Journal of Immunological Methods, 95 (1986) 177-186 Elsevier
177
JIM 04160
Specific IgE antibodies to reactive dye-albumin conjugates C.M. Luczynska and M.D. Topping Occupational Medicine and Hygiene Laboratories, Health and Safety Executive, London, U.K. (Received 6 May 1986, accepted 21 August 1986)
Hypersensitivity to reactive dye powders has been recognised for a number of years, although the extent of sensitisation amongst dye house operatives and the immunochemistry of the dye molecules has not been investigated. We have developed a radioallergosorbent test (RAST) to detect specific IgE to reactive dye-human serum albumin (HSA) conjugates. From a total of 19 dye-HSA conjugates, positive RASTs were found in six workers with allergic symptoms associated with dye exposure, while six asymptomatic case-matched controls were negative. Sera with raised total IgE (up to 4300 kU/l) from unexposed workers gave negative results except for two conjugates which gave a weak positive at 4300 kU/1 and one which gave weak positives at all concentrations tested (750-4300 kU/1). RAST inhibition studies demonstrated that the antibody was specific for the complete dye-HSA conjugate. Substitution of bovine serum albumin (BSA) for HSA in the conjugate mdrkedly reduced immunoreactivity and free hapten gave lower inhibition than the complete conjugate. Comparison of the dye-HSA RAST with a RAST using dyed discs showed that the latter did not correlate well with symptoms and was influenced by the total IgE concentration. Key words: IgE; Reactive dye powder; Radioallergosorbent test; (Antibodies)
Introduction Low molecular weight chemicals, capable of reaction with a human serum protein, have the potential to act as allergens through the formation of a hapten-carrier conjugate. An association between allergic respiratory symptoms and IgE antibodies has been shown for only a few low molecular weight chemicals. These include the acid
Correspondence to: M.D. Topping, Occupational Medicine and Hygiene Laboratories, Health and Safety Executive, 403 Edgware Road, London NW2 6LN, U.K. Abbreviations: IEA, immunoelectrophoretic analysis; RAST, radioallergosorbent test; PBS, phosphate-buffered saline pH 7.4; cpm, counts per minute; PRIST, paper-radioimmunosorbent test; HSA, human serum albumin; BSA, bovine serum albumin; C.I., Colour Index; CV, coefficient of variation.
anhydrides, phthalic anhydride (PA) (Maccia et al., 1976), trimellitic anhydride (TMA) (Zeiss et al., 1982) and tetrachlorophthalic anhydride (TCPA) (Howe et al., 1983). Specific IgE antibodies to ethylene oxide have been demonstrated (Marshall et al., 1985) in patients with anaphylactic reactions after haemodialysis. Also, IgE antibodies to quaternary or tertiary ammonium ions on succinyl choline, decamethonium and gallamine muscle relaxants have been detected in anaphylactic patients (Harle et al., 1985). Since their introduction in 1956, reactive dyes have been used extensively in the textile industry. The advantages of reactive dyes over other types of dye are that a lower dyeing temperature and better colour fastness can be achieved. In 1978 Alanko et al. described four workers with immediate-type hypersensitivity to reactive dye
178
powders, in whom skin prick and provocation testing with dye powders gave positive results. Kalas and Runstukova (1980) found that over a quarter of 106 textile workers investigated were sensitised to reactive dyes and 7% had symptoms suggesting occupational asthma. Reactive dyes have molecular weights ranging from 500 to 1000. They contain a chromogen linked to a reactive functional group designed to form irreversible covalent bonds with hydroxyl groups on cellulose fibres. Fig. 1 shows the structures of four of the dyes included in this study (Colour Index, 1982). The Procion reactive dyes shown have a dichlorotriazine functional group, reacting by nucleophilic substitution and the Remazol reactive dye has a/3-sulphatoethyl sulphone group, a precursor of vinyl sulphone, which reacts by nucleophilic addition onto the unsaturated
Procion
Red MX5B
/N% OH
Procion
NH %
CI /N
Yellow MX4R
~=--'~ N~OH H03S--( | ])"l II
S(~3H ~
~Cl N~%
co o. Procion Orange
carbon bond (Elliot and Yeung, 1979). Other reactive systems commonly used include monochlorotriazine, monochlorodifluoropyrimidine and dichloropyridazinone as well as vinyl sulphone. These have the same basic reaction mechanism but are less reactive (Rattee, 1969). The reaction of dichlorotriazine dyes with amino acids has also been investigated (Shore, 1968a). The main functional group of proteins reacting with reactive dyes are the E-amino group of lysine, the imidazole group of histidine and thiol group of cysteine. Which of these three main types of reaction will predominate depends on the pH, amino acid composition and molecular weight of the protein. Studies with TMA suggest that the target protein in low molecular weight chemical haptenprotein complexes is human serum albumin (HSA) (Zeiss et al., 1977). HSA contains 59 lysines, 16 histidines and one free cysteinyl thiol group (Meloun et al., 1975). It can be assumed that reactive dyes are capable of covalent linkage to these groups in human serum albumin by the same mechanism by which they combine to cellulose fibres (Shore, 1968b). With the reactive dye systems based on nucleophilic substitution, this involves the reaction mechanism outlined in Fig. 2. After an increase in the use of reactive dyes since the 1960s, immediate-type sensitisation of dye house workers to different dyes was reported (Alanko et al., 1978). A radioallergosorbeot test (RAST) to detect specific IgE antibodies to reac-
MX2R
SO3H
CI
OH N=N
N
DYE CHROMOGEN -- N H ' ~
03 SO3H
N
~CI
)
+ NH2CH2etc
CI
RemazolBlackB HO
NH2
DYE CHROMOGEN -- NH"~'--~//N
.\ NaO3s" g
g
+
H ~ Cl-
Leaving group
"SO3Na Dye-human serum albumin conjugate
R =
aminoterminal of lysineof HSA
I
Dichlorotriazine reactive group
SO2CH2CH2OSO3Na
Fig. 1. Structures of some of the reactive dyes used in study.
etc
Fig. 2. Proposed reaction between a dichlorotriazine dye and h u m a n serum albumin.
179
Materials and methods
(PBS), with two changes, for 24 h at room temperature. The reactive dye-HSA conjugates were stored at - 2 0 ° C . Paper discs (589/3, Schleicher and Schull, F.R.G.) were activated with cyanogen bromide (Ceska et al., 1972). 600 mg activated discs were tumble-mixed with 1.5 ml reactive dyeHSA conjugate or 20 m g / m l HSA and 15 ml 0.1 M sodium bicarbonate (8.4 g/l) overnight at room temperature. The discs were washed four times with PBS and stored in RAST disc storage buffer (Pharmacia, Uppsala, Sweden) at 4°C. Reactive dye-HSA conjugates at different ratios were prepared using Procion Red MX5B. 2.5, 5, 10, 15, 25, 35 and 50 mg were mixed with 5 ml HSA as before to give reaction mixtures with molar ratios of 3, 6, 12, 18, 30, 50 and 60:1 reactive d y e : H S A . These were dialysed against several changes of PBS until no more free dye was present in the dialysate. Discs were prepared using the different ratio dye-HSA conjugates as previously described.
Subjects
Preparation of other conjugate discs
Dye house operatives working with a wide range of reactive dyes, differing in both reactive system and colour, were used as the study group. Blood samples were obtained from six individuals ( A - F ) with respiratory symptoms associated with exposure to reactive dyes. Appropriate controls case-matched for age, length of exposure, and smoking ( A C - F C ) were chosen from workers exposed to the same dyes but who had no symptoms. Each subject was tested with the dyes to which he was exposed. Samples of dyes were obtained from the dye houses; a total of 19 were used in this study. Sera from unexposed subjects with a high total IgE concentration were also tested.
Reactive dye-bovine serum albumin (BSA) conjugates were prepared as before, substituting BSA (98-99%, A7030, Sigma) for HSA. TCPA-HSA conjugates were prepared as previously described (Howe et al., 1983).
tive dyes was carried out using paper discs dyed under the same conditions as cellulose fibres. However, a RAST in which the reactive dye is coupled to a human serum protein is more likely to reflect the chemical conjugate formed in vivo if the dye molecules act as haptens. This paper reports the development and validation of a RAST to detect specific IgE to reactive dye-human serum albumin conjugates in subjects with allergic respiratory symptoms associated with exposure to reactive dye powders. The effect of total IgE on the RAST was investigated. RAST inhibition studies were carried out to determine the sensitivity and specificity of the assay. The RAST results for symptomatic and unexposed subjects with high total IgE using reactive dye-human serum albumin conjugates were compared with those obtained using dyed paper discs.
Preparation of reactive dye-HSA conjugate discs 5 mg of reactive dye was mixed with 5 ml of 20 m g / m l HSA (crystallised, 96-99% albumin, A9511, Sigma) in 50 mM carbonate/bicarbonate buffer, pH 10 (!.56 g sodium carbonate, 2.92 g sodium bicarbonate/l). This reaction mixture gave a reactive dye : HSA molar ratio of approximately 6 : 1 , assuming an average reactive dye molecular weight of 600. The reactive dye-HSA mixture was dialysed against 2 litre phosphate-buffered saline
Preparation of dyed discs Dyed discs were prepared by a method similar to that described by (Alanko et al., 1978). Unactivated paper discs were washed in 50 g/1 sodium chloride. 50 mg of reactive dye powder were dissolved in 10 ml of a saline/carbonate solution (50 g/1 NaC1, 20 g/1 sodium carbonate) and rotated with 250 mg washed discs overnight at room temperature. The dyed discs were washed extensively in cold running water until no more free dye washed off and the discs stored at 4°C in RAST disc storage buffer (Pharmacia).
Characterisation of reactive dye-HSA conjugates Immunoelectrophoretic analysis. Evidence for conjugation was obtained using immunoelectrophoretic analysis (IEA) in 1.5% Agarose-M (LKB, 2206-101) in 0.02 M barbiturate buffer, pH 8.6.5 /~1 of the conjugates were used and electrophoresis was carried out at 25 mA, 220 V until the HSA-
180 b r o m o p h e n o l blue marker had migrated 4 cm. The gels were developed with 50/zl r a b b i t a n t i - h u m a n a l b u m i n ( A l 1 9 , D A K O , C o p e n h a g e n ) overnight at 4 ° C. Speetrophotometrie analysis. Spectrophotometry was carried out to estimate the p r o p o r t i o n of reactive dye b o u n d to HSA. The a b s o r b a n c e of d y e - H S A conjugates, before a n d after dialysis, was measured at the wavelength of m a x i m u m a b s o r b a n c e of the dye c h r o m o g e n (Table I). The a m o u n t of dye coupled to a l b u m i n was calculated from the change in a b s o r b a n c e d u r i n g dialysis. Molecular weights are n o t published for a n u m b e r of the dyes, therefore results for dye b i n d i n g were expressed as mg d y e / / ~ m o l of HSA. For the Procion Red M X 5 B - H S A conjugates of different ratios prepared, the molar b i n d i n g ratio could be calculated since the molecular weight was known.
H S A disc, overnight at room temperature. The discs were washed three times with 1.5 ml of 0.9% saline c o n t a i n i n g R A S T washing solution additive (Pharmacia). 50/~1 of a25I-labelled rabbit anti-hum a n IgE (Pharmacia) a n d 50 #1 PBS were added a n d left overnight at r o o m temperature. The discs were washed again three times with washing solution a n d the b o u n d 125I measured using a g a m m a counter. All the assays were performed in duplicate. The results were expressed as R A S T percent b i n d i n g , defined as percentage of added counts per m i n u t e (cpm) b o u n d to the reactive d y e - H S A conjugate disc m i n u s the percentage of added c p m b o u n d to the H S A disc.
Measurement of total IgE Total IgE was measured using a Phadebas P R I S T kit (Pharmacia) according to the m a n u f a c turer's instructions.
Radioallergosorbent test 200 #1 of serum, diluted 1 in 4 in PBS, were i n c u b a t e d with a reactive d y e - H S A conjugate disc a n d a further 200 /zl of diluted serum with an
RA S T inhibition A serum with high specific IgE, diluted 1 in 10 in PBS, was mixed with 100 ~1 of PBS or 100 ktl
TABLE I CHARACTERISATION OF DYE-HSA CONJUGATES USING SPECTROMETRY AND IMMUNOELECTROPHORESIS Dye-HSA conjugate
Cibacron Red RB Levafix Red E5BN Procion Red MXG Procion Red MX5B Procion Red MX7B Cibacron Blue FGF Cibacron Blue FR Procion Blue MXG Cibacron Navy FG Procion Navy HER Procion Navy MXPBK Cibaeron Yellow F3R Levafix Yellow E2RA Levafix Yellow E3RL Procion Yellow MX3R Procion Yellow MX4R Procion Orange MX2R Procion Brown MX5BR Remazol Black B a
~, max
Spectrometry: dye/HSA mix A max
dye-HSA conj A max
550 525 515 520 550 605 600 620 640 610 615 430 425 400 420 445 490 530 595
1.288 0.763 0.999 0.966 0.905 1.025 1.514 0.664 0.933 0.924 0.805 1.144 0.810 0.881 0.699 0.980 1.243 0.583 1.209
0.932 0.578 0.597 0.594 0.793 0.998 1.181 0.467 0.710 0.710 0.617 0.786 0.559 0.721 0.528 0.755 0.953 0.464 0.909
Results corrected for volume change after dialysis.
mg d y e / ~mol HSA a
IEA: mobility relative to HSA
2.3 2.5 2.2 2.0 2.9 3.5 2.4 2.3 2.5 2.4 2.8 2.2 2.3 2.8 2.3 2.6 2.5 2.9 2.5
1.21 1.15 1.03 1.14 1.16 1.16 1.22 1.03 1.32 1.03 1.14 1.27 1.20 1.17 1.10 1.23 1.15 1.11 1.14
181 aliquots of inhibitor diluted in PBS. A reactive dye-HSA conjugate disc was added and the tubes incubated overnight at room temperature. All inhibitions were carried out in duplicate. 100 /zl of diluted serum and 100/~1 PBS were also incubated together with an HSA disc, also in duplicate. IgE bound to the discs was measured as described for specific IgE. The mean counts bound to the HSA disc were subtracted from the mean counts bound to the reactive dye-HSA conjugate discs and the results expressed as percentage inhibition, calculated as follows: bound to dye-HSAdisc with inhibitor ) 100-( cpmcpmbound to dye-HSAdisc without inhibitor × 100
Hydrolysis of reactive dyes Reactive dyes with reactive groups inactivated by hydrolysis were required for the RAST hapten inhibition studies and were prepared by dissolving 5 mg dye powder in 5 ml 50 mM carbonate/bicarbonate buffer, pH 10 and leaving the solutions at 40°C overnight to hydrolyse. In order to check that hydrolysis had occurred the dye hydrolysates were mixed with HSA and dialysed. Some dye absorbed onto the albumin and lEA was carried out on the retentate to evaluate whether conjugation had occurred. Before use in RAST inhibition the dye hydrolysates were diluted 1 in 2 with 0.2 M phosphate buffer, pH 7.5 (5 g KH2PO 4, 22 g Na2HPO4/1 ) to reduce the p H to 8. This phosphate buffer was used to make the inhibitor dilutions and appropriate controls.
Results
Characterisation of reactive dye-HSA conjugates Immunoelectrophoretic analysis. The conjugation of reactive dye with HSA was easily verified. Virtually no free dye could be seen outside the dialysis bag compared with reactive dye solutions, and the retained reactive dye-HSA conjugate was highly coloured, lEA of the reactive dye-HSA conjugates showed increased mobility, compared to HSA, towards the positive terminal, indicating that the net negative charge of the albumin molecule had been increased on binding to the reactive dye. Table I shows the electrophoretic mobilities,
relative to albumin, for each of the reactive dyeHSA conjugates. Spectrophotometric analysis. Mixing of some of the dyes with HSA causes a small change in the wavelength at which the dye chromogen absorbs maximally (bathochromic shift), independent of the amount of dye present. In order to overcome this, the calculations to estimate the extent of dye binding to albumin are based on the absorbance of dye albumin reaction mixture, in carbonate/bicarbonate buffer, pH 10. The HSA present in the reactive dye conjugates does not contribute to the absorbance of dye chromogen which can therefore be attributed totally to the amount of reactive dye present. Table I shows the amount of reactive dye coupled to albumin for each of the reactive dyeHSA conjugates. For the Procion Red MX5B-HSA conjugates, the Beer-Lambert Law was used to calculate the molar binding ratios for the 3, 6, 12, 18, 30, 50 and 60:1 Procion Red M X 5 B : H S A molar reaction mixtures, and these were 3, 4, 5, 8, 9, 18 and 28 : 1 Procion Red MX5B to HSA, respectively.
Direct RAST % binding in symptomatic and asymptomatic subjects RAST results were expressed as RAST % binding, after subtraction of the % binding to the HSA disc. Net RAST % binding of more than 0.4% was taken to be indicative of the presence of specific IgE. This value was the mean control serum % binding plus 2.5 times the standard deviation of the mean control % serum binding. The mean % binding to the HSA discs was 0.5%, with an intra-assay CV of 10% and inter-assay CV, 15%. The results for the symptomatic dye workers are shown in Table II, together with the total IgE concentration. Three individuals (B, D and E) had positive RASTs to all the reactive dyes to which they were exposed but only associated their symptoms with some of the dyes. The remaining symptomatic operatives had positive RASTs to at least a quarter of the dyes to which they were exposed. Where no result is shown the subject was not in contact, and therefore not tested, with the particular dye concerned. The case-matched asymptomatic controls were tested with the same dyes as the symptomatic worker with which they were matched and all were negative.
182 TABLE II DIRECT RAST % BINDING TO DYE-HSA CONJUGATES Dye-HSA conjugate
RAST % binding A
Cibacron Red RB Levafix Red E5BN Procion Red MXG Procion Red MX5B Procion Red MX7B Cibacron Blue FGF Cibacron Blue FR Procion Blue MXG Cibacron Navy FG Procion Navy HER Procion Navy MXPBK Cibacron Yellow F3R Levafix Yellow E2RA Levafix Yellow E3RL Procion Yellow MX3R Procion Yellow MX4R Procion Orange MX2R Procion Brown MX5BR Remazol Black B Total IgE (kU/1)
B
C
D
E
F
NT 4.2 0.5 1.8 0.5 NT 1.4 6.6 NT 11.1 NT 12.7 7.7 NT 3.5 1.8
1.2 NT a 17.7 3.4 5.8 NT NT 0.8 0.9 NT 10.3 4.1 NT 1.1 4.6 1.6 4.8 9.2 NT
0.6 0.2 0.6 0.0 0.2 -0.1 0.0 0.0 0.1 2.0 NT 1.4 2.0 NT 0.5 0.9 1.0 0.0 NT
1.9 NT NT NT NT 0.5 NT NT 2.2 NT NT 5.0 NT NT NT NT NT NT 0.9
17.2 NT 3.3 17.7 NT 2.0 7.9 1.7 10.8 NT 12.1 6.5 NT 2.8 20.2 NT NT 2.7 NT
1.0 NT NT NT NT 0.1 0.6 0.2 0.1 0.1 NT 0.3 NT NT NT NT NT NT 0.7
2410
474
251
383
54
2.0 2.0 3.1
76
a Not tested; not exposed to dye.
Direct R A S T % binding in sera with raised total IgE R A S T s with reactive d y e - H S A conjugate discs a n d three u n e x p o s e d sera with increasingly high c o n c e n t r a t i o n s of total IgE showed that 'false positives' were only o b t a i n e d at the highest IgE c o n c e n t r a t i o n tested (4300 k U / l ) for Procion Blue M X G a n d Procion N a v y H E R (0.4% b i n d i n g ) a n d at c o n c e n t r a t i o n s above 748 k U / 1 for Remazol Black B (0.5% binding). The relatively large size of the Remazol Black B dye c h r o m o g e n ( M W 993, Fig. 1) m a y a c c o u n t for the non-specific b i n d i n g of IgE to these d y e - H S A conjugate discs. The b i n d i n g to H S A discs was also more p r o n o u n c e d with increasing levels of total IgE.
Effect of reactive dye : HSA ratio on R A S T % binding The molar b i n d i n g ratio of Procion Red M X 5 B - H S A increased in p r o p o r t i o n to the a m o u n t of dye added, perhaps i n d i c a t i n g that the b i n d i n g capacity of H S A for the dye had n o t been exceeded. T a b l e III shows the R A S T results for two
s y m p t o m a t i c a n d two a s y m p t o m a t i c (exposed) workers a n d three sera with raised total IgE. A t b i n d i n g ratios greater t h a n 9 : 1 'false positive' results were o b t a i n e d with the sera having high total IgE c o n c e n t r a t i o n s (748 k U / 1 a n d above). At the c o n j u g a t i o n ratio used in the study (approximately 4 : 1, based o n an average molecular weight of 600), 'false positives' were not o b t a i n e d below a total IgE c o n c e n t r a t i o n of 4300 k U / 1 .
Comparison of reactive dye-HSA conjugate discs with dyed discs in symptomatic subjects and controls The R A S T results o b t a i n e d for three symptomatic a n d three a s y m p t o m a t i c individuals using reactive d y e - H S A conjugate discs a n d dyed discs were c o m p a r e d for four dyes: Procion Red MX5B, Procion Red M X G , Procion Yellow M X 3 R a n d C i b a c r o n Yellow F3R. The reactive d y e - H S A conj u g a t e % b i n d i n g s were corrected for H S A b i n d i n g a n d the % b i n d i n g to a n undyed, unactivated p a p e r disc was subtracted from the % b i n d i n g to the dyed discs. The results are shown in T a b l e IV. T h e dyed disc R A S T s did not distinguish between
183
TABLE III E F F E C T OF R E A C T I V E D Y E : HSA RATIO ON RAST % B I N D I N G TO R E A C T I V E DYE-HSA DISCS Ratio of Procion Red MX5B : HSA
% Binding B b
3:1 4:1 5:1 8:1 9:1 18:1 28:1 TotallgE (kU/1) a b c 0
2.2 2.3 3.8 3.9 3.6 2.0 1.9
474
BC c 0.2 -0.1 -0.1 -0.1 0.0 -0.1 0.0
175
E b 22.7 21.6 26.9 28.7 27.8 25.1 22.2
383
EC ~" 0.0 0.1 0.0 0.0 0.1 0.1 0.2
382
E1 d 0.0 0.0 0.1 0.1 0.5 0.8 1.6
748
E2 d NT a 0.0 0.1 0.0 0.8 1.1 NT
2090
E3 d 0.2 0.4 0.4 0.3 1.3 2.1 3.9
4300
N o t tested. Symptomatic subjects. Controls. Three sera with a raised total IgE level.
symptomatic and asymptomatic exposed individuals whereas the dye-HSA conjugate RAST results correlated well with the presence of symptoms.
Comparison of reactive dye-HSA conjugate discs with dyed discs: effect of high total lgE Table V shows the results for two symptomatic, two asymptomatic and six unexposed subjects with
high serum IgE for dyed discs and reactive dyeHSA conjugate discs, together with the total IgE concentration. The dyed disc % binding appeared to reflect the total IgE concentration rather than the presence of specific antibody. With unexposed individuals the RAST % binding (also corrected for blank disc binding) correlated well with total IgE.
T A B L E IV D I R E C T RAST C O M P A R I S O N OF R E A C T I V E DYE-HSA C O N J U G A T E DISCS W I T H D Y E D DISCS IN S Y M P T O M A T I C A N D A S Y M P T O M A T I C SUBJECTS Subjects B a
C a
D y e - H S A conjugate discs: Procion Red MX5B Procion Red M X G Procion Yellow MX3 R Cibacron Yellow F3 R
3.4 17.7 4.6 4.1
0.0 0.6 0.5 1.4
D y e d discs: Procion Red MX5B Procion Red M X G Procion Yellow MX3 R Cibacron Yellow F3 R
2.1 3.4 2.1 14.3
2.0 1.3 2.5 14.0
Total IgE (kU/l) a Symptomatic subjects. b Controls.
474
76
E a
BC b
CC b
EC b
17.7 3.3 20.2 6.5
0.0 0.2 -0.1 0.1
0.1 0.2 0.1 0.0
0.1 0.3 0.3 0.0
4.4 3.9 3.5 17.0
1.8 1.2 3.1 12.0
1.5 1.6 2.1 16.5
1.6 1.0 1.3 12.1
383
175
105
382
184 TABLE V DIRECT RAST COMPARISON OF REACTIVE DYE-HSA CONJUGATE DISCS WITH DYED DISCS: EFFECT OF HIGH TOTAL IgE Group
Total IgE (kU/l)
°c 15
% Binding LevafixYellow E2RA
Symptomatic 2410 474 Asymptomatic 204 Controls 54
16.2 8.6 2.9 3.0
11.1 3.8 0.1 0.0
G H I J K L
Sera of unexposed individuals
4.5 5.5 6.4 13.1 10.9 15.4
0.0 0.0 0.1 0.3 0.4 0.2
303 500 640 1020 1641 6538
~ +
70 80
}--..
\......
-:
90 100
3
10
30 /~g Inhibitor
~
9o
°°F
a
~
1~
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I 30 #g Inhibitor
#glnhibitoredded
3100
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1~00
300
-
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1O0 added
~o~-
RA S T inhibition Hydrolysed dyes, prepared for i n h i b i t i o n experiments as free haptens, were mixed with HSA, dialysed, a n d electrophoresed. T h e result showed that the mobility of H S A had n o t been altered b y i n c u b a t i o n with the reactive dye hydrolysates, showing that the reactive group had been inactivated b y the hydrolysis procedure. Fig. 3a a n d b shows the R A S T % i n h i b i t i o n o b t a i n e d for various inhibitors using serum from subject A a n d Levafix Yellow E 2 R A - H S A discs a n d Procion Yellow M X 3 R - H S A discs. Fig. 3c a n d d shows the R A S T i n h i b i t i o n data for s e r u m from subject E a n d Procion Red M X 5 B - H S A discs a n d Procion
0o *
40
~ so ~. 6o
Dyed discs Dye-HSA conjugate on discs A B AC EC
-
3O
I 100
"~..~ I I ~'" 300 1000
added
Fig. 3. RAST inhibition of dye specific IgE by dye-HSA conjugate (O), dye hydrolysate (11), dye-BSA (A), TCPA-HSA (O) and HSA (+) (expressed as ~g carrier added): (a) subject AS, dye and discs Levafix Yellow E2RA-HSA, uninhibited % binding: 7.6%; (b) subject AS, dye and discs Procion Yellow MX3R-HSA, uninhibited % binding: 7.2%; (c) subject ES, dye and discs Procion Yellow MX3R-HSA, uninhibited % binding: 5.5%; (d) subject ES, dye and discs Procion Red MX5B-HSA uninhibited % binding: 5.6%.
185 Yellow MX3R-HSA discs. The reactive dye-HSA conjugate is in all cases the most effective inhibitor, reaching maximum inhibition at 300 /~g inhibitor added. Altering the carrier by substituting BSA for HSA has the effect of decreasing the amount of inhibition elicited at the same concentration. Free reactive dye hydrolysates were more effective inhibitors than reactive dye-BSA conjugates but not as strong as the reactive dyeHSA conjugates. Neither carrier alone (HSA) nor an unrelated hapten (TCPA) coupled to HSA gave any significant inhibition. Reactive dye-HSA conjugates did not inhibit an unrelated hapten RAST system and the binding of TCPA induced specific IgE to TCPA-HSA conjugate discs.
Discussion
We have shown that reactive dyes can act as haptens. Combination with human serum albumin can be achieved in vitro and specific IgE antibodies to dye-HSA conjugates has been demonstrated in individuals with respiratory tract symptoms associated with exposure to these dyes. RAST inhibition studies using different haptencarrier combinations demonstrated the specificity of the RAST. Highest inhibition was obtained with the complete conjugate, but the free reactive dye hydrolysates gave considerable % inhibition as haptens suggesting that the antibody is mainly directed towards the hapten portion of the conjugate. Alternatively, this high hapten inhibition could be due to the free dye being passively absorbed onto HSA in the serum during incubation, and forming a non-covalent conjugate capable of inhibiting in the RAST. Reactive dye-BSA conjugates gave lower inhibition showing that carrier specificity is also important in the recognition of the reactive dye conjugate by the antibody. HSA alone and TCPA-HSA gave no inhibition, confirming the role of the hapten in the antibody combining site. When the reactive dye conjugates were used to inhibit a TCPA-HSA RAST, no inhibition of the unrelated RAST system was obtained, showing that the inhibition found with the conjugate is not due to non-specific factors. The RAST % binding cut-off value of 0.4%, based on the mean control % binding plus 2.5 standard
deviations, is similar to that reported in a previous study using TCPA-HSA (Venables et al., 1985). However, the value would need to be reassessed in any subsequent study using a larger population of different workers. In the preparation of reactive dye: HSA conjugates of differing ratios, virtually no free dye was dialysed away in the low ratio conjugates. Immunoelectrophoretic analysis showed that the amount of reactive dye bound to albumin increased with the amount of reactive dye added. In symptomatic individuals the greatest reactivity in the RAST was at a reactive dye-HSA ratio of 8:1. This ratio is similar to that found during ratio studies using TCPA-HSA conjugates (Topping et al., 1986). Comparison of the RAST results for dyed discs with reactive dye-HSA conjugates on discs showed that the RAST % binding for an unconjugated reactive dye system reflected the total IgE concentration and was therefore not a reliable measure of specific IgE. This is possibly due to the large number of dye molecules bound to the paper disc acting as an ion exchanger for the IgE. In contrast, a high total IgE concentration of less than 4000 kU/1 does not interfere with the reactive dye-HSA conjugate RAST we have developed, providing that binding to HSA discs is subtracted. The RAST using dyed discs gave results that did not correlate well with workers' symptoms, whereas the reactive dye-HSA RAST was able to detect specific IgE in only those individuals who had symptoms associated with exposure to reactive dye powders. In a previous comparison of the two RASTs using 30 workers from a textile factory a good correlation was found between dyed disc % binding and total IgE (r = 0.79, P < 0.0005), whereas the correlation between reactive dye-HSA conjugate % binding and total IgE was not significant (r = 0.25, P > 0.25). Further data on the relationship between the reactive dye-HSA conjugate RAST and work-related symptoms is at present being assessed. A number of individuals with antibodies to dye conjugates had positive RASTs to reactive dyes with which they did not associate their symptoms. As some of the reactive dyes share the same chromogen and differ only in the individual reactive system, it is likely that some immunological cross reactivity is present between some of the reactive
186
dye-HSA conjugates. Studies currently being undertaken are expected to provide information on the immunochemistry of reactive dyes. In conclusion, we have found that specific IgE to reactive dye-HSA conjugates correlates well with allergic symptoms associated with exposure to dyes, providing strong evidence that allergy to these dyes is mediated by IgE antibody. A RAST using dye-HSA conjugates is a more physiologically relevant test than that using dyed discs where we found interference from total IgE. RAST inhibition data demonstrated that the RAST is specific for the reactive dye-HSA conjugate and is not affected by an unrelated hapten system.
Acknowledgements We would like to thank our clinical colleagues, Dr. J. Wattie, Dr. A. Docker, Dr. A.J. Newman Taylor and Dr. C.A. Pickering, for blood samples from their patients.
References Alanko, K., H. Keskinen, F. Bjorksten and S. Ojanen, 1978, Clin. Allergy 8, 25.
Ceska, M., R. Eriksonn and J.M. Vargo, 1972, J. Allergy Clin. Immunol. 49, 1. Colour Index, 1982, 3rd edn. (2nd rev.) Society of Dyers and Colourers. Elliot, J. and P.P. Yeung, 1979, Encyclopaedia of Chemical Technology, 3rd edn., Vol. 8, p. 374. Harle, D.G., B.A. Baldo and M.M. Fisher, 1985, J. Immunol. Methods 78, 293. Howe, W., K.M. Venables, M.D. Topping, M.B. Dally, R. Hawkins, J.S. Law and A.J. Newman Taylor, 1983, J. Allergy Clin. Immunol. 71, 5. Kalas, D. and J. Runstukova, 1980, Prac. Lek. 32, 103. Maccia, C.A., I.L. Bernstein, E.A. Emmett and S.M. Brooks, 1976, Am. Rev. Respir. Dis. 113, 701. Marshall, C.P., F.C. Pearson, M.A. Sagona, W. Lee, R.L. Wathen, R.A. Ward and J. Dolovich, 1985, J. Allergy Clin. Immunol. 75, 563. Meloun, B., L. Moravek and V. Kostka, 1975, FEBS Lett. 58, 134. Rattee, I.D., 1969, J. Soc. Dyers Colour. 85, 23. Shore, J., 1968a, J. Soc. Dyers Colour. 84, 545. Shore, J., 1968b, J. Soc. Dyers Colour. 84, 413. Topping, M.D., K.M. Venables, C.M. Luczynska, W. Howe and A.J. Newman Taylor, 1986, J. Allergy Clin. Immunol. 77, 834. Venables, K.M., M.D. Topping, W. Howe, C.M. Luczynska, R. Hawkins and A.J. Newman Taylor, 1985, Br. Med. J. 290, 201. Zeiss, C.R., R. Patterson, J.J. Pruzansky, M.M. Miller, M. Rosenberg and D. Levitz, 1977, J. Allergy Clin. Immunol. 60, 96. Zeiss, C.R., P. Wolkonsky, J.J. Pruzansky and R. Patterson, 1982, J. Allergy Clin. Immunol. 70, 15.
177
JIM 04160
Specific IgE antibodies to reactive dye-albumin conjugates C.M. Luczynska and M.D. Topping Occupational Medicine and Hygiene Laboratories, Health and Safety Executive, London, U.K. (Received 6 May 1986, accepted 21 August 1986)
Hypersensitivity to reactive dye powders has been recognised for a number of years, although the extent of sensitisation amongst dye house operatives and the immunochemistry of the dye molecules has not been investigated. We have developed a radioallergosorbent test (RAST) to detect specific IgE to reactive dye-human serum albumin (HSA) conjugates. From a total of 19 dye-HSA conjugates, positive RASTs were found in six workers with allergic symptoms associated with dye exposure, while six asymptomatic case-matched controls were negative. Sera with raised total IgE (up to 4300 kU/l) from unexposed workers gave negative results except for two conjugates which gave a weak positive at 4300 kU/1 and one which gave weak positives at all concentrations tested (750-4300 kU/1). RAST inhibition studies demonstrated that the antibody was specific for the complete dye-HSA conjugate. Substitution of bovine serum albumin (BSA) for HSA in the conjugate mdrkedly reduced immunoreactivity and free hapten gave lower inhibition than the complete conjugate. Comparison of the dye-HSA RAST with a RAST using dyed discs showed that the latter did not correlate well with symptoms and was influenced by the total IgE concentration. Key words: IgE; Reactive dye powder; Radioallergosorbent test; (Antibodies)
Introduction Low molecular weight chemicals, capable of reaction with a human serum protein, have the potential to act as allergens through the formation of a hapten-carrier conjugate. An association between allergic respiratory symptoms and IgE antibodies has been shown for only a few low molecular weight chemicals. These include the acid
Correspondence to: M.D. Topping, Occupational Medicine and Hygiene Laboratories, Health and Safety Executive, 403 Edgware Road, London NW2 6LN, U.K. Abbreviations: IEA, immunoelectrophoretic analysis; RAST, radioallergosorbent test; PBS, phosphate-buffered saline pH 7.4; cpm, counts per minute; PRIST, paper-radioimmunosorbent test; HSA, human serum albumin; BSA, bovine serum albumin; C.I., Colour Index; CV, coefficient of variation.
anhydrides, phthalic anhydride (PA) (Maccia et al., 1976), trimellitic anhydride (TMA) (Zeiss et al., 1982) and tetrachlorophthalic anhydride (TCPA) (Howe et al., 1983). Specific IgE antibodies to ethylene oxide have been demonstrated (Marshall et al., 1985) in patients with anaphylactic reactions after haemodialysis. Also, IgE antibodies to quaternary or tertiary ammonium ions on succinyl choline, decamethonium and gallamine muscle relaxants have been detected in anaphylactic patients (Harle et al., 1985). Since their introduction in 1956, reactive dyes have been used extensively in the textile industry. The advantages of reactive dyes over other types of dye are that a lower dyeing temperature and better colour fastness can be achieved. In 1978 Alanko et al. described four workers with immediate-type hypersensitivity to reactive dye
178
powders, in whom skin prick and provocation testing with dye powders gave positive results. Kalas and Runstukova (1980) found that over a quarter of 106 textile workers investigated were sensitised to reactive dyes and 7% had symptoms suggesting occupational asthma. Reactive dyes have molecular weights ranging from 500 to 1000. They contain a chromogen linked to a reactive functional group designed to form irreversible covalent bonds with hydroxyl groups on cellulose fibres. Fig. 1 shows the structures of four of the dyes included in this study (Colour Index, 1982). The Procion reactive dyes shown have a dichlorotriazine functional group, reacting by nucleophilic substitution and the Remazol reactive dye has a/3-sulphatoethyl sulphone group, a precursor of vinyl sulphone, which reacts by nucleophilic addition onto the unsaturated
Procion
Red MX5B
/N% OH
Procion
NH %
CI /N
Yellow MX4R
~=--'~ N~OH H03S--( | ])"l II
S(~3H ~
~Cl N~%
co o. Procion Orange
carbon bond (Elliot and Yeung, 1979). Other reactive systems commonly used include monochlorotriazine, monochlorodifluoropyrimidine and dichloropyridazinone as well as vinyl sulphone. These have the same basic reaction mechanism but are less reactive (Rattee, 1969). The reaction of dichlorotriazine dyes with amino acids has also been investigated (Shore, 1968a). The main functional group of proteins reacting with reactive dyes are the E-amino group of lysine, the imidazole group of histidine and thiol group of cysteine. Which of these three main types of reaction will predominate depends on the pH, amino acid composition and molecular weight of the protein. Studies with TMA suggest that the target protein in low molecular weight chemical haptenprotein complexes is human serum albumin (HSA) (Zeiss et al., 1977). HSA contains 59 lysines, 16 histidines and one free cysteinyl thiol group (Meloun et al., 1975). It can be assumed that reactive dyes are capable of covalent linkage to these groups in human serum albumin by the same mechanism by which they combine to cellulose fibres (Shore, 1968b). With the reactive dye systems based on nucleophilic substitution, this involves the reaction mechanism outlined in Fig. 2. After an increase in the use of reactive dyes since the 1960s, immediate-type sensitisation of dye house workers to different dyes was reported (Alanko et al., 1978). A radioallergosorbeot test (RAST) to detect specific IgE antibodies to reac-
MX2R
SO3H
CI
OH N=N
N
DYE CHROMOGEN -- N H ' ~
03 SO3H
N
~CI
)
+ NH2CH2etc
CI
RemazolBlackB HO
NH2
DYE CHROMOGEN -- NH"~'--~//N
.\ NaO3s" g
g
+
H ~ Cl-
Leaving group
"SO3Na Dye-human serum albumin conjugate
R =
aminoterminal of lysineof HSA
I
Dichlorotriazine reactive group
SO2CH2CH2OSO3Na
Fig. 1. Structures of some of the reactive dyes used in study.
etc
Fig. 2. Proposed reaction between a dichlorotriazine dye and h u m a n serum albumin.
179
Materials and methods
(PBS), with two changes, for 24 h at room temperature. The reactive dye-HSA conjugates were stored at - 2 0 ° C . Paper discs (589/3, Schleicher and Schull, F.R.G.) were activated with cyanogen bromide (Ceska et al., 1972). 600 mg activated discs were tumble-mixed with 1.5 ml reactive dyeHSA conjugate or 20 m g / m l HSA and 15 ml 0.1 M sodium bicarbonate (8.4 g/l) overnight at room temperature. The discs were washed four times with PBS and stored in RAST disc storage buffer (Pharmacia, Uppsala, Sweden) at 4°C. Reactive dye-HSA conjugates at different ratios were prepared using Procion Red MX5B. 2.5, 5, 10, 15, 25, 35 and 50 mg were mixed with 5 ml HSA as before to give reaction mixtures with molar ratios of 3, 6, 12, 18, 30, 50 and 60:1 reactive d y e : H S A . These were dialysed against several changes of PBS until no more free dye was present in the dialysate. Discs were prepared using the different ratio dye-HSA conjugates as previously described.
Subjects
Preparation of other conjugate discs
Dye house operatives working with a wide range of reactive dyes, differing in both reactive system and colour, were used as the study group. Blood samples were obtained from six individuals ( A - F ) with respiratory symptoms associated with exposure to reactive dyes. Appropriate controls case-matched for age, length of exposure, and smoking ( A C - F C ) were chosen from workers exposed to the same dyes but who had no symptoms. Each subject was tested with the dyes to which he was exposed. Samples of dyes were obtained from the dye houses; a total of 19 were used in this study. Sera from unexposed subjects with a high total IgE concentration were also tested.
Reactive dye-bovine serum albumin (BSA) conjugates were prepared as before, substituting BSA (98-99%, A7030, Sigma) for HSA. TCPA-HSA conjugates were prepared as previously described (Howe et al., 1983).
tive dyes was carried out using paper discs dyed under the same conditions as cellulose fibres. However, a RAST in which the reactive dye is coupled to a human serum protein is more likely to reflect the chemical conjugate formed in vivo if the dye molecules act as haptens. This paper reports the development and validation of a RAST to detect specific IgE to reactive dye-human serum albumin conjugates in subjects with allergic respiratory symptoms associated with exposure to reactive dye powders. The effect of total IgE on the RAST was investigated. RAST inhibition studies were carried out to determine the sensitivity and specificity of the assay. The RAST results for symptomatic and unexposed subjects with high total IgE using reactive dye-human serum albumin conjugates were compared with those obtained using dyed paper discs.
Preparation of reactive dye-HSA conjugate discs 5 mg of reactive dye was mixed with 5 ml of 20 m g / m l HSA (crystallised, 96-99% albumin, A9511, Sigma) in 50 mM carbonate/bicarbonate buffer, pH 10 (!.56 g sodium carbonate, 2.92 g sodium bicarbonate/l). This reaction mixture gave a reactive dye : HSA molar ratio of approximately 6 : 1 , assuming an average reactive dye molecular weight of 600. The reactive dye-HSA mixture was dialysed against 2 litre phosphate-buffered saline
Preparation of dyed discs Dyed discs were prepared by a method similar to that described by (Alanko et al., 1978). Unactivated paper discs were washed in 50 g/1 sodium chloride. 50 mg of reactive dye powder were dissolved in 10 ml of a saline/carbonate solution (50 g/1 NaC1, 20 g/1 sodium carbonate) and rotated with 250 mg washed discs overnight at room temperature. The dyed discs were washed extensively in cold running water until no more free dye washed off and the discs stored at 4°C in RAST disc storage buffer (Pharmacia).
Characterisation of reactive dye-HSA conjugates Immunoelectrophoretic analysis. Evidence for conjugation was obtained using immunoelectrophoretic analysis (IEA) in 1.5% Agarose-M (LKB, 2206-101) in 0.02 M barbiturate buffer, pH 8.6.5 /~1 of the conjugates were used and electrophoresis was carried out at 25 mA, 220 V until the HSA-
180 b r o m o p h e n o l blue marker had migrated 4 cm. The gels were developed with 50/zl r a b b i t a n t i - h u m a n a l b u m i n ( A l 1 9 , D A K O , C o p e n h a g e n ) overnight at 4 ° C. Speetrophotometrie analysis. Spectrophotometry was carried out to estimate the p r o p o r t i o n of reactive dye b o u n d to HSA. The a b s o r b a n c e of d y e - H S A conjugates, before a n d after dialysis, was measured at the wavelength of m a x i m u m a b s o r b a n c e of the dye c h r o m o g e n (Table I). The a m o u n t of dye coupled to a l b u m i n was calculated from the change in a b s o r b a n c e d u r i n g dialysis. Molecular weights are n o t published for a n u m b e r of the dyes, therefore results for dye b i n d i n g were expressed as mg d y e / / ~ m o l of HSA. For the Procion Red M X 5 B - H S A conjugates of different ratios prepared, the molar b i n d i n g ratio could be calculated since the molecular weight was known.
H S A disc, overnight at room temperature. The discs were washed three times with 1.5 ml of 0.9% saline c o n t a i n i n g R A S T washing solution additive (Pharmacia). 50/~1 of a25I-labelled rabbit anti-hum a n IgE (Pharmacia) a n d 50 #1 PBS were added a n d left overnight at r o o m temperature. The discs were washed again three times with washing solution a n d the b o u n d 125I measured using a g a m m a counter. All the assays were performed in duplicate. The results were expressed as R A S T percent b i n d i n g , defined as percentage of added counts per m i n u t e (cpm) b o u n d to the reactive d y e - H S A conjugate disc m i n u s the percentage of added c p m b o u n d to the H S A disc.
Measurement of total IgE Total IgE was measured using a Phadebas P R I S T kit (Pharmacia) according to the m a n u f a c turer's instructions.
Radioallergosorbent test 200 #1 of serum, diluted 1 in 4 in PBS, were i n c u b a t e d with a reactive d y e - H S A conjugate disc a n d a further 200 /zl of diluted serum with an
RA S T inhibition A serum with high specific IgE, diluted 1 in 10 in PBS, was mixed with 100 ~1 of PBS or 100 ktl
TABLE I CHARACTERISATION OF DYE-HSA CONJUGATES USING SPECTROMETRY AND IMMUNOELECTROPHORESIS Dye-HSA conjugate
Cibacron Red RB Levafix Red E5BN Procion Red MXG Procion Red MX5B Procion Red MX7B Cibacron Blue FGF Cibacron Blue FR Procion Blue MXG Cibacron Navy FG Procion Navy HER Procion Navy MXPBK Cibaeron Yellow F3R Levafix Yellow E2RA Levafix Yellow E3RL Procion Yellow MX3R Procion Yellow MX4R Procion Orange MX2R Procion Brown MX5BR Remazol Black B a
~, max
Spectrometry: dye/HSA mix A max
dye-HSA conj A max
550 525 515 520 550 605 600 620 640 610 615 430 425 400 420 445 490 530 595
1.288 0.763 0.999 0.966 0.905 1.025 1.514 0.664 0.933 0.924 0.805 1.144 0.810 0.881 0.699 0.980 1.243 0.583 1.209
0.932 0.578 0.597 0.594 0.793 0.998 1.181 0.467 0.710 0.710 0.617 0.786 0.559 0.721 0.528 0.755 0.953 0.464 0.909
Results corrected for volume change after dialysis.
mg d y e / ~mol HSA a
IEA: mobility relative to HSA
2.3 2.5 2.2 2.0 2.9 3.5 2.4 2.3 2.5 2.4 2.8 2.2 2.3 2.8 2.3 2.6 2.5 2.9 2.5
1.21 1.15 1.03 1.14 1.16 1.16 1.22 1.03 1.32 1.03 1.14 1.27 1.20 1.17 1.10 1.23 1.15 1.11 1.14
181 aliquots of inhibitor diluted in PBS. A reactive dye-HSA conjugate disc was added and the tubes incubated overnight at room temperature. All inhibitions were carried out in duplicate. 100 /zl of diluted serum and 100/~1 PBS were also incubated together with an HSA disc, also in duplicate. IgE bound to the discs was measured as described for specific IgE. The mean counts bound to the HSA disc were subtracted from the mean counts bound to the reactive dye-HSA conjugate discs and the results expressed as percentage inhibition, calculated as follows: bound to dye-HSAdisc with inhibitor ) 100-( cpmcpmbound to dye-HSAdisc without inhibitor × 100
Hydrolysis of reactive dyes Reactive dyes with reactive groups inactivated by hydrolysis were required for the RAST hapten inhibition studies and were prepared by dissolving 5 mg dye powder in 5 ml 50 mM carbonate/bicarbonate buffer, pH 10 and leaving the solutions at 40°C overnight to hydrolyse. In order to check that hydrolysis had occurred the dye hydrolysates were mixed with HSA and dialysed. Some dye absorbed onto the albumin and lEA was carried out on the retentate to evaluate whether conjugation had occurred. Before use in RAST inhibition the dye hydrolysates were diluted 1 in 2 with 0.2 M phosphate buffer, pH 7.5 (5 g KH2PO 4, 22 g Na2HPO4/1 ) to reduce the p H to 8. This phosphate buffer was used to make the inhibitor dilutions and appropriate controls.
Results
Characterisation of reactive dye-HSA conjugates Immunoelectrophoretic analysis. The conjugation of reactive dye with HSA was easily verified. Virtually no free dye could be seen outside the dialysis bag compared with reactive dye solutions, and the retained reactive dye-HSA conjugate was highly coloured, lEA of the reactive dye-HSA conjugates showed increased mobility, compared to HSA, towards the positive terminal, indicating that the net negative charge of the albumin molecule had been increased on binding to the reactive dye. Table I shows the electrophoretic mobilities,
relative to albumin, for each of the reactive dyeHSA conjugates. Spectrophotometric analysis. Mixing of some of the dyes with HSA causes a small change in the wavelength at which the dye chromogen absorbs maximally (bathochromic shift), independent of the amount of dye present. In order to overcome this, the calculations to estimate the extent of dye binding to albumin are based on the absorbance of dye albumin reaction mixture, in carbonate/bicarbonate buffer, pH 10. The HSA present in the reactive dye conjugates does not contribute to the absorbance of dye chromogen which can therefore be attributed totally to the amount of reactive dye present. Table I shows the amount of reactive dye coupled to albumin for each of the reactive dyeHSA conjugates. For the Procion Red MX5B-HSA conjugates, the Beer-Lambert Law was used to calculate the molar binding ratios for the 3, 6, 12, 18, 30, 50 and 60:1 Procion Red M X 5 B : H S A molar reaction mixtures, and these were 3, 4, 5, 8, 9, 18 and 28 : 1 Procion Red MX5B to HSA, respectively.
Direct RAST % binding in symptomatic and asymptomatic subjects RAST results were expressed as RAST % binding, after subtraction of the % binding to the HSA disc. Net RAST % binding of more than 0.4% was taken to be indicative of the presence of specific IgE. This value was the mean control serum % binding plus 2.5 times the standard deviation of the mean control % serum binding. The mean % binding to the HSA discs was 0.5%, with an intra-assay CV of 10% and inter-assay CV, 15%. The results for the symptomatic dye workers are shown in Table II, together with the total IgE concentration. Three individuals (B, D and E) had positive RASTs to all the reactive dyes to which they were exposed but only associated their symptoms with some of the dyes. The remaining symptomatic operatives had positive RASTs to at least a quarter of the dyes to which they were exposed. Where no result is shown the subject was not in contact, and therefore not tested, with the particular dye concerned. The case-matched asymptomatic controls were tested with the same dyes as the symptomatic worker with which they were matched and all were negative.
182 TABLE II DIRECT RAST % BINDING TO DYE-HSA CONJUGATES Dye-HSA conjugate
RAST % binding A
Cibacron Red RB Levafix Red E5BN Procion Red MXG Procion Red MX5B Procion Red MX7B Cibacron Blue FGF Cibacron Blue FR Procion Blue MXG Cibacron Navy FG Procion Navy HER Procion Navy MXPBK Cibacron Yellow F3R Levafix Yellow E2RA Levafix Yellow E3RL Procion Yellow MX3R Procion Yellow MX4R Procion Orange MX2R Procion Brown MX5BR Remazol Black B Total IgE (kU/1)
B
C
D
E
F
NT 4.2 0.5 1.8 0.5 NT 1.4 6.6 NT 11.1 NT 12.7 7.7 NT 3.5 1.8
1.2 NT a 17.7 3.4 5.8 NT NT 0.8 0.9 NT 10.3 4.1 NT 1.1 4.6 1.6 4.8 9.2 NT
0.6 0.2 0.6 0.0 0.2 -0.1 0.0 0.0 0.1 2.0 NT 1.4 2.0 NT 0.5 0.9 1.0 0.0 NT
1.9 NT NT NT NT 0.5 NT NT 2.2 NT NT 5.0 NT NT NT NT NT NT 0.9
17.2 NT 3.3 17.7 NT 2.0 7.9 1.7 10.8 NT 12.1 6.5 NT 2.8 20.2 NT NT 2.7 NT
1.0 NT NT NT NT 0.1 0.6 0.2 0.1 0.1 NT 0.3 NT NT NT NT NT NT 0.7
2410
474
251
383
54
2.0 2.0 3.1
76
a Not tested; not exposed to dye.
Direct R A S T % binding in sera with raised total IgE R A S T s with reactive d y e - H S A conjugate discs a n d three u n e x p o s e d sera with increasingly high c o n c e n t r a t i o n s of total IgE showed that 'false positives' were only o b t a i n e d at the highest IgE c o n c e n t r a t i o n tested (4300 k U / l ) for Procion Blue M X G a n d Procion N a v y H E R (0.4% b i n d i n g ) a n d at c o n c e n t r a t i o n s above 748 k U / 1 for Remazol Black B (0.5% binding). The relatively large size of the Remazol Black B dye c h r o m o g e n ( M W 993, Fig. 1) m a y a c c o u n t for the non-specific b i n d i n g of IgE to these d y e - H S A conjugate discs. The b i n d i n g to H S A discs was also more p r o n o u n c e d with increasing levels of total IgE.
Effect of reactive dye : HSA ratio on R A S T % binding The molar b i n d i n g ratio of Procion Red M X 5 B - H S A increased in p r o p o r t i o n to the a m o u n t of dye added, perhaps i n d i c a t i n g that the b i n d i n g capacity of H S A for the dye had n o t been exceeded. T a b l e III shows the R A S T results for two
s y m p t o m a t i c a n d two a s y m p t o m a t i c (exposed) workers a n d three sera with raised total IgE. A t b i n d i n g ratios greater t h a n 9 : 1 'false positive' results were o b t a i n e d with the sera having high total IgE c o n c e n t r a t i o n s (748 k U / 1 a n d above). At the c o n j u g a t i o n ratio used in the study (approximately 4 : 1, based o n an average molecular weight of 600), 'false positives' were not o b t a i n e d below a total IgE c o n c e n t r a t i o n of 4300 k U / 1 .
Comparison of reactive dye-HSA conjugate discs with dyed discs in symptomatic subjects and controls The R A S T results o b t a i n e d for three symptomatic a n d three a s y m p t o m a t i c individuals using reactive d y e - H S A conjugate discs a n d dyed discs were c o m p a r e d for four dyes: Procion Red MX5B, Procion Red M X G , Procion Yellow M X 3 R a n d C i b a c r o n Yellow F3R. The reactive d y e - H S A conj u g a t e % b i n d i n g s were corrected for H S A b i n d i n g a n d the % b i n d i n g to a n undyed, unactivated p a p e r disc was subtracted from the % b i n d i n g to the dyed discs. The results are shown in T a b l e IV. T h e dyed disc R A S T s did not distinguish between
183
TABLE III E F F E C T OF R E A C T I V E D Y E : HSA RATIO ON RAST % B I N D I N G TO R E A C T I V E DYE-HSA DISCS Ratio of Procion Red MX5B : HSA
% Binding B b
3:1 4:1 5:1 8:1 9:1 18:1 28:1 TotallgE (kU/1) a b c 0
2.2 2.3 3.8 3.9 3.6 2.0 1.9
474
BC c 0.2 -0.1 -0.1 -0.1 0.0 -0.1 0.0
175
E b 22.7 21.6 26.9 28.7 27.8 25.1 22.2
383
EC ~" 0.0 0.1 0.0 0.0 0.1 0.1 0.2
382
E1 d 0.0 0.0 0.1 0.1 0.5 0.8 1.6
748
E2 d NT a 0.0 0.1 0.0 0.8 1.1 NT
2090
E3 d 0.2 0.4 0.4 0.3 1.3 2.1 3.9
4300
N o t tested. Symptomatic subjects. Controls. Three sera with a raised total IgE level.
symptomatic and asymptomatic exposed individuals whereas the dye-HSA conjugate RAST results correlated well with the presence of symptoms.
Comparison of reactive dye-HSA conjugate discs with dyed discs: effect of high total lgE Table V shows the results for two symptomatic, two asymptomatic and six unexposed subjects with
high serum IgE for dyed discs and reactive dyeHSA conjugate discs, together with the total IgE concentration. The dyed disc % binding appeared to reflect the total IgE concentration rather than the presence of specific antibody. With unexposed individuals the RAST % binding (also corrected for blank disc binding) correlated well with total IgE.
T A B L E IV D I R E C T RAST C O M P A R I S O N OF R E A C T I V E DYE-HSA C O N J U G A T E DISCS W I T H D Y E D DISCS IN S Y M P T O M A T I C A N D A S Y M P T O M A T I C SUBJECTS Subjects B a
C a
D y e - H S A conjugate discs: Procion Red MX5B Procion Red M X G Procion Yellow MX3 R Cibacron Yellow F3 R
3.4 17.7 4.6 4.1
0.0 0.6 0.5 1.4
D y e d discs: Procion Red MX5B Procion Red M X G Procion Yellow MX3 R Cibacron Yellow F3 R
2.1 3.4 2.1 14.3
2.0 1.3 2.5 14.0
Total IgE (kU/l) a Symptomatic subjects. b Controls.
474
76
E a
BC b
CC b
EC b
17.7 3.3 20.2 6.5
0.0 0.2 -0.1 0.1
0.1 0.2 0.1 0.0
0.1 0.3 0.3 0.0
4.4 3.9 3.5 17.0
1.8 1.2 3.1 12.0
1.5 1.6 2.1 16.5
1.6 1.0 1.3 12.1
383
175
105
382
184 TABLE V DIRECT RAST COMPARISON OF REACTIVE DYE-HSA CONJUGATE DISCS WITH DYED DISCS: EFFECT OF HIGH TOTAL IgE Group
Total IgE (kU/l)
°c 15
% Binding LevafixYellow E2RA
Symptomatic 2410 474 Asymptomatic 204 Controls 54
16.2 8.6 2.9 3.0
11.1 3.8 0.1 0.0
G H I J K L
Sera of unexposed individuals
4.5 5.5 6.4 13.1 10.9 15.4
0.0 0.0 0.1 0.3 0.4 0.2
303 500 640 1020 1641 6538
~ +
70 80
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-:
90 100
3
10
30 /~g Inhibitor
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a
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I 30 #g Inhibitor
#glnhibitoredded
3100
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40 ~
I
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0
I
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OI
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1~00
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-
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100
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RA S T inhibition Hydrolysed dyes, prepared for i n h i b i t i o n experiments as free haptens, were mixed with HSA, dialysed, a n d electrophoresed. T h e result showed that the mobility of H S A had n o t been altered b y i n c u b a t i o n with the reactive dye hydrolysates, showing that the reactive group had been inactivated b y the hydrolysis procedure. Fig. 3a a n d b shows the R A S T % i n h i b i t i o n o b t a i n e d for various inhibitors using serum from subject A a n d Levafix Yellow E 2 R A - H S A discs a n d Procion Yellow M X 3 R - H S A discs. Fig. 3c a n d d shows the R A S T i n h i b i t i o n data for s e r u m from subject E a n d Procion Red M X 5 B - H S A discs a n d Procion
0o *
40
~ so ~. 6o
Dyed discs Dye-HSA conjugate on discs A B AC EC
-
3O
I 100
"~..~ I I ~'" 300 1000
added
Fig. 3. RAST inhibition of dye specific IgE by dye-HSA conjugate (O), dye hydrolysate (11), dye-BSA (A), TCPA-HSA (O) and HSA (+) (expressed as ~g carrier added): (a) subject AS, dye and discs Levafix Yellow E2RA-HSA, uninhibited % binding: 7.6%; (b) subject AS, dye and discs Procion Yellow MX3R-HSA, uninhibited % binding: 7.2%; (c) subject ES, dye and discs Procion Yellow MX3R-HSA, uninhibited % binding: 5.5%; (d) subject ES, dye and discs Procion Red MX5B-HSA uninhibited % binding: 5.6%.
185 Yellow MX3R-HSA discs. The reactive dye-HSA conjugate is in all cases the most effective inhibitor, reaching maximum inhibition at 300 /~g inhibitor added. Altering the carrier by substituting BSA for HSA has the effect of decreasing the amount of inhibition elicited at the same concentration. Free reactive dye hydrolysates were more effective inhibitors than reactive dye-BSA conjugates but not as strong as the reactive dyeHSA conjugates. Neither carrier alone (HSA) nor an unrelated hapten (TCPA) coupled to HSA gave any significant inhibition. Reactive dye-HSA conjugates did not inhibit an unrelated hapten RAST system and the binding of TCPA induced specific IgE to TCPA-HSA conjugate discs.
Discussion
We have shown that reactive dyes can act as haptens. Combination with human serum albumin can be achieved in vitro and specific IgE antibodies to dye-HSA conjugates has been demonstrated in individuals with respiratory tract symptoms associated with exposure to these dyes. RAST inhibition studies using different haptencarrier combinations demonstrated the specificity of the RAST. Highest inhibition was obtained with the complete conjugate, but the free reactive dye hydrolysates gave considerable % inhibition as haptens suggesting that the antibody is mainly directed towards the hapten portion of the conjugate. Alternatively, this high hapten inhibition could be due to the free dye being passively absorbed onto HSA in the serum during incubation, and forming a non-covalent conjugate capable of inhibiting in the RAST. Reactive dye-BSA conjugates gave lower inhibition showing that carrier specificity is also important in the recognition of the reactive dye conjugate by the antibody. HSA alone and TCPA-HSA gave no inhibition, confirming the role of the hapten in the antibody combining site. When the reactive dye conjugates were used to inhibit a TCPA-HSA RAST, no inhibition of the unrelated RAST system was obtained, showing that the inhibition found with the conjugate is not due to non-specific factors. The RAST % binding cut-off value of 0.4%, based on the mean control % binding plus 2.5 standard
deviations, is similar to that reported in a previous study using TCPA-HSA (Venables et al., 1985). However, the value would need to be reassessed in any subsequent study using a larger population of different workers. In the preparation of reactive dye: HSA conjugates of differing ratios, virtually no free dye was dialysed away in the low ratio conjugates. Immunoelectrophoretic analysis showed that the amount of reactive dye bound to albumin increased with the amount of reactive dye added. In symptomatic individuals the greatest reactivity in the RAST was at a reactive dye-HSA ratio of 8:1. This ratio is similar to that found during ratio studies using TCPA-HSA conjugates (Topping et al., 1986). Comparison of the RAST results for dyed discs with reactive dye-HSA conjugates on discs showed that the RAST % binding for an unconjugated reactive dye system reflected the total IgE concentration and was therefore not a reliable measure of specific IgE. This is possibly due to the large number of dye molecules bound to the paper disc acting as an ion exchanger for the IgE. In contrast, a high total IgE concentration of less than 4000 kU/1 does not interfere with the reactive dye-HSA conjugate RAST we have developed, providing that binding to HSA discs is subtracted. The RAST using dyed discs gave results that did not correlate well with workers' symptoms, whereas the reactive dye-HSA RAST was able to detect specific IgE in only those individuals who had symptoms associated with exposure to reactive dye powders. In a previous comparison of the two RASTs using 30 workers from a textile factory a good correlation was found between dyed disc % binding and total IgE (r = 0.79, P < 0.0005), whereas the correlation between reactive dye-HSA conjugate % binding and total IgE was not significant (r = 0.25, P > 0.25). Further data on the relationship between the reactive dye-HSA conjugate RAST and work-related symptoms is at present being assessed. A number of individuals with antibodies to dye conjugates had positive RASTs to reactive dyes with which they did not associate their symptoms. As some of the reactive dyes share the same chromogen and differ only in the individual reactive system, it is likely that some immunological cross reactivity is present between some of the reactive
186
dye-HSA conjugates. Studies currently being undertaken are expected to provide information on the immunochemistry of reactive dyes. In conclusion, we have found that specific IgE to reactive dye-HSA conjugates correlates well with allergic symptoms associated with exposure to dyes, providing strong evidence that allergy to these dyes is mediated by IgE antibody. A RAST using dye-HSA conjugates is a more physiologically relevant test than that using dyed discs where we found interference from total IgE. RAST inhibition data demonstrated that the RAST is specific for the reactive dye-HSA conjugate and is not affected by an unrelated hapten system.
Acknowledgements We would like to thank our clinical colleagues, Dr. J. Wattie, Dr. A. Docker, Dr. A.J. Newman Taylor and Dr. C.A. Pickering, for blood samples from their patients.
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