Use of immunoblot technique for detection of human IgE and IgG antibodies to individual silk proteins

Use of immunoblot technique for detection human IgE and IgG antibodies to individual silk proteins Mahmoud Munich

Dewair,

Ph.D.,* Xaver Baur, M.D.,* and Klaus Ziegler,

and Aachen, Federal Republic

of

Ph.D.**

of Germany

Allergenic proteins were extracted from one silk batch that was imported to be used as jlling material for bed mattresses and rugs. IgE and IgG antibodies to the extracted silk proteins were measured by RAST in sera of nine silk-sensitive persons as well as in sera of healthy control donors. Silk proteins were fractionated by sodium dodecyl suljate-polyacrylamide gel electrophoresis into 12 polypeptides of molecular weights between 14 and 70 kilodaltons. By means of the immunoblot technique, IgE and IgG antibodies to the individual silk polypeptides could be detected. Sera of silk-sensitive persons contained high titers of IgE and low titers of IgG antibodies to the separated silk polypeptides. Sera of control donors contained low IgG antibody titers to a limited number of these polypeptides. (J ALLERGY CLIN IM,WUNCN 76:53742, 1985.)

It has been reported that allergic bronchial asthma can be caused by environmental’ or occupational’ exposure to silkworms, silk, or silk wastes. Reaginic sensitivity to wild silk is also known.‘. ’ This sensitivity is induced either during employment in silk factories or after acquiring rugs filled with silk wastes. Silk” is produced by silkworms (Bombyx mori Lint@) that feed on mulberry leaves, whereas wild silk, from which tussah silk is obtained, is produced by wild growing worms (Antheraea mylitta, A. perni, or A. yamamai) that live on oak trees. Either kind of silk (mulberry-worm silk or oak-worm silk) is made of two main components: fibroin, a fibrous protein that makes up the core of the silk filament, and sericin, a less well-characterized protein(s) that surrounds the spun fiber and functions as an adhesive. Sericin is also known as silk gum. The sericin is usually removed from silk during manufacture in the so-called degumming process in which silk is washed in alkaline soap baths. The degummed intact silk fibers are used as high-quality silk, whereas incomplete and interrupted fibers make the silk wastes that are usually used as cheap filling for bed mattresses and rugs. According to the method of Fuchs,3 sericin is the

From the *F%eumologischeAbteilung, Medizinische Klinik I, Klinikum Grosshadem, University of Munich. and **Deutsches Wollforschungsinstitut,Aachen, FederalRepublic of Germany. Supportedby DeutscheForschungsgemeinschaft. Received for publication April 9, 1984. Accepted for publication Dec. 21. 1984. Reprint requestsare not available.

Abbreviations used

PMSF: Phenylmethanesulfonyl fluoride SP: Silk proteins

HS-PBST: 50 mmol/L Na2HP0,/NaHZP0,, pH 7.5, 150 mmol/L NaCl, 0.04% NaN,. 1% Tween 20, 2% horse serum PBST: SDS: NC:

Same as HS-PBST but lacking the horse serum

Sodium dodecyl sulfate Nitrocellulose membrane

PBS-O.1T: 50 mmol/L Na,HPO,/NaH,PO,, 150 mmol/L NaCl, 0.1% Tween 20 kd: kilodaltons PRU: PhadebasRAST unit PCS:

Pooled control serum

only pathogenic antigen, since he tested six different wild silk extracts and found that the more se&in present in the extract the stronger was the skin test response to it. Sericin culture-related asthma, review by Kobayashi,’ is mainly induced by exposure to the urine of silkworm larvae and scales of silkworm moths, whereas sericin and silk threads themselves have only weak allergenic activity. Similar to this finding are the findings of Kino and Oshima’ that silkworm wings, butterflies, and moths should contain the offending allergens in the case of asthma caused by environmental exposure to silkworms. We present in this article the application of the immunoblot technique6 for characterization of individual allergenic and antigenic polypeptides in one 537

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et al.

I. Means of exposure

TABLE

to silk and clinical

No.

1 2 3 4 5 6 7 8 9

Sex

(yr)

F F M F M F M M M

35 21 45 30 58 26 23 32 42

Serum No. 1

2 3 4 5 6 7 8 9 PCS

IgE RAST” (PRUlml)

IgG RASTt (cpmlpaper disc)

24.1 21.1 24.0 25.9 25.3 23.8 24.6 12.3 - 8.9

756 703 1063 1022 660 843 634 831 759

0.05

595

*PRU: a unit used to measurespecific IgE antibody titers in test serumsamplesrelative to one positive referenceserumincluded in PhadebasRAST kit (PhannaciaDiagnostics, AB). Test serum sampleseliciting 0.35 PRU/ml are consideredpositive. tIgG RAST values for sera from nine silk-sensitive patients, Nos. 1 to 9, and for PCS.

silk extract. By use of sera obtained from nine persons allergic to silk, five polypeptides were found that can be considered as the main allergens in this extract. Several other minor allergens could also be detected. All the sera of allergic subjects investigated, and to a lesser extent a PCS, were found to contain IgG antibodies to some polypeptides in the extract. MATERIAL Sera

AND METHODS

Test sera were collected from nine persons allergic to silk or to rugs filled with different kinds of silk or silk wastes. All these persons had been suffering from bronchial asthma and rhinitis; five of them also developed conjunctivitis after contact with the silk material. The sensitization occurred after exposure to silk products or to silk-filled rugs. Skin tests by use of one silk extract elicited positive results with all nine persons; wheal-and-flare reactions comparable to those produced by 1 mg/ml of histamine solution occurred

patients Clinical

User Producer User Producer User User Producer User User

II. IgE RAST and IgG RAST values

TABLE

of silk-allergic

Means of exposure to silk rugs

Age Patient

diagnoses

CLIN. IMMUNOL. OCTOBER 1985

Asthma, Asthma, Asthma Asthma, Asthma, Asthma, Asthma, Asthma Asthma,

diagnoses

rhinitis rhinitis, conjunctivitis rhinitis, conjunctivitis rhinitis rhinitis, conjunctivitis rhinitis rhinitis, conjunctivitis

in all patients. Several control persons had negative results when they were skin tested with this extract. A more detailed description of the nine patients whose sera were used in this study is illustrated in Table I. PCS was prepared from 2 1 serum samples obtained from healthy donors free of respiratory symptoms; in some ex-

periments serum samples from individual control donors were used. Silk extracts Silk extracts were prepared from four silk batches imported from China, India, and Brazil to be used as filling for mattresses and rugs. Each extract was made by shaking 100 gm of the silk material in 5 L of buffer A (1 mol/L NaCI, 100 mmol/L NaHCO,, and 1 mmol/L PMSF) for 20 hours at room temperature. The buffer was then collected by decanting and pressing the silk residue. This extraction was repeated once more, and the extracts were pooled. Ammonium sulfate was added to the extract to a final saturation of 90% and stirred for 16 hours. The precipitated material was collected by centrifugation, then dissolved in and dialyzed against water, and finally was lyophilized. The protein content was determined according to the method of Lowry et aL7 The yield was about 30 mg of SP. The allergenic activities of the four extracts were tested by RAST, and the one active extract, prepared from one of the two Chinese silk batches, was further used for skin tests, RASTs and immunoblots.

Binding

of SP to paper discs

SP were bound to CNBr-activated paper discs as described by Ceska et al.* by use of 5 pg of SP per paper disc.

Determination

of IgE antibodies

to SP

Antibodies were measured by the IgE RAST (Phadebas RAST kit, Pharmacia Diagnostics, AB, Uppsala, Sweden) by use of the SP paper discs described above.

Determination

of IgG antibodies

to SP

Antibodies were measured by use of a modification’ of the IgG RAST described by Shimizu et al.” Briefly, each

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lmmunoblot

technique

for IgE and IgG antibodies

539

FIG. 1. IgG PAST values of sera from nine patients (P.S.) and sera from 21 control donors (C’S). SP were bound to paper discs. The paper discs were incubated with human sera, washed, incubated with ‘ZSl-anti-human IgG, and washed again. The remaining bound radioactivity was measured in a gamma counter.

SP paper disc was incubated overnight in 100 pl of human serum diluted 1: 400 in HS-PBST. Each paper disc was then washed three times, each time by incubation for 1 hour in 2 ml of PBST. Discs were then incubated for 2 hours, each in 50 p.1 of “‘I-labeled goat anti-human IgG of specific activity about 5 to 7 pCi/pg (New England Nuclear, Boston, Mass.) that was diluted to 90 nglml in HS-PBST and then washed as before. Bound radioactivity was then measured in a gamma counter.

Polyacrylamide immunoblotting

gel electrophoresis

and

Slabs of 7% to 18% exponential gradient polyacrylamide gels were used for the electrophoretic separation of SP. Electrophoresis was carried out in a discontinuous buffer system containing 0.1% SDS”; 3.5 mg of SP dissolved in the sample buffer” was applied onto the whole gel by use of a l-well comb. At the end of the electrophoretic run, two 1.5 cm marginal strips were cut out from the gel, fixed in trichloroacetic acid, and stained in Coomassie brilliant blue R-250. The remaining middle part of the gel was brought into contact with a nitrocellulose sheet (Bio-Rad Laboratories, Richmond, Calif.), and proteins were electrophoretically transferred to the NC as described by Towbin et a1.6After the transfer, a 0.5 cm wide marginal strip was cut from the NC sheet and stained with amido black. The rest of the NC sheet was incubated twice in 200 ml of PBS0.1 T.‘?. I3 For each serum sample, two 0.5 cm NC strips were used, one for detection of specific IgE and the other for detection of specific IgG antibodies. For detection of IgE antibodies, the strip was incubated overnight with human serum diluted 1: 5, washed, incubated with 16 ng of ‘ZSI-labeled rabbit anti-human IgE immunoglobulin (Phadebas, Pharmacia), washed, and then auto-

FIG. 2. SDS-gradient polyacrylamide gel electrophoresis. Lane at molecular weight marker polypeptides. Lane br silk polypeptides.

radiographed. For detection of specific IgG antibodies, the NC strip was incubated in human serum diluted 1: 100, washed, incubated in ‘251-labeled goat anti-human IgG immunoglobulin (New England Nuclear), washed, and then autoradiographed. Washing of NC strips was carried out by shaking in PBS-O.1 T, and the same buffer was used for dilution of human sera and radioactive reagents. Autoradiography was carried out by putting the dried NC strips in contact with Kodak XAR (Eastman Kodak Co., Rochester, N.Y.) films for varying lengths of time.

RESULTS Four batches of commercially available silk wastes that were industrially prepared to be used as filling for mattresses and rugs were extracted. Two of the batches used were of Chinese origin; the other two came from India and Brazil. The extract of one of the two Chinese silk samples elicited high IgE RAST results as well as positive skin tests, whereas extracts made from the other three batches elicited negative results. Proteins extracted from this allergen-containing silk sample were further used in all the RAST and immunoblot experiments described. This silk sample was chemically and microscopically analysed at the German Institute of Wool Research (Aachen, Federal

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FIG. 3. lmmunoblot illustrating IgE antibodies to individual silk polypeptides in sera of nine silksensitive patients (lanes 7 to 9) and in PCS (lane 10). The electrophoretically resolved polypeptides were transferred to NC. The NC was cut into strips. Each strip was incubated with one serum, washed, incubated with ‘Z51-anti-human IgE, washed again, and was autoradiographed.

Republic of Germany). According to the results of these analyses, the material was identified as Bombyx mori silk, consisting mainly of fibroin with little sericin content. The content of sericin in this batch was, however, higher than in the other three batches. The results of IgE RAST are illustrated in Table II. All nine test sera demonstrated relatively high IgE RAST titers, whereas the PCS produced essentially a background RAST value. Several serum samples from individual control donors elicited RAST values similar to that of PCS, and hence, these values were not included. It was of interest to know whether sera from silk-sensitive persons also contained anti-SP IgG antibodies. To answer this question, an IgG RAST was used that was carried out under conditions modified from those published by others.” The modifications introduced in this article (see Material and Methods) were found to differentiate between positive and negative serum sample by a factor of more than 10 as compared with another envi-

ronmental antigen, Aspergillus fumigatus, and sera from patients with bronchopulmonary aspergillosis.’ This IgG RAST was carried out by use of the SP paper discs with sera from the nine silk-sensitive persons and sera from 21 control donors as well as the PCS. The results are presented in Table II and Fig. 1. Data in Fig. 1 suggest that the IgG RAST values of sera from patients and control donors are comparable. Statistical analysis by use of the t test, however, demonstrated a significant difference between the two mean IgG RAST values for sera from patients and control donors (p < 0.01). By use of Kendall’s statistical method, no significant correlation could be detected between the IgE and IgG RAST values for the sera from nine patients (7 = 0.036). These RAST results were then confirmed and extended by the immunoblotting technique. SF could be resolved by polyacrylamide gel elec-

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trophoresis into 12 polypeptides of molecular weights between 14 and 70 kd (Fig. 2). One of these polypeptides of molecular weight, 40 kd, is found in much greater quantity than the others. The resolved polypeptides were electrophoretically transferred to an NC that was cut into strips and incubated with sera of different patients, and one strip was also incubated with PCS. rZ51-anti-human IgE and “%-anti-human IgG were used to detect human IgE and IgG anti-SP antibodies, respectively. Fig. 3 illustrates the distribution in sera from patients (srrips 1 to 9) and in PCS (strip 10) of IgE antibodies to individual SP. Four polypeptides appear to represent the major allergens in SP; these are polypeptides of molecular weights about 70,45, 40, and 14 kd. Binding of “‘l-anti-human IgE to these polypeptides was found in the serum of eight of the nine patients tested. IgE antibodies to minor polypeptides also exist. Serum from different patients appears to have similar but not identical spectra of IgE antibodies to the individual SP. For example, IgE antibody to the 40 kd polypeptide was completely absent in patient serum No. 2, and patient serum No. 5 contained no IgE antibodies to most of the minor polypeptides. PCS demonstrated no binding of IgE antibodies at all, and thus, this strip was virtually identical to the blank ones. Fig. 4 illustrates the distribution of IgG antibodies to different SP in sera from patients and in the PCS. IgG antibodies to two polypeptides of molecular weights 70 and 6 kd were found in all 10 sera examined including the PCS. In addition, patients’ sera contained antibodies to the major 40 kd polypeptide. Comparison of Figs. 3 and 4 illustrate that at least three polypeptides (of about 70, 45, and 40 kd molecular weights) bind both IgE and IgG antibodies, whereas one polypeptide of molecular weight about 6 kd bound only IgG.

DISCUSSION Silk contains potent allergenic proteins that can be extracted under mild conditions. Four silk batches were extracted, and the allergenic activities of the extracts were screened by RAST. The one extract found to elicit high IgE RAST titers and positive skin tests was used further in this study and was made from one silk batch that was imported to be used as filling material for rugs. All patients’ sera demonstrated high IgE anti-SP antibody titers ranging from 9 to more than 25 PRU/ ml compared to less than 0.05 PRU/ml with the PCS. IgG RAST values, however, did not distinguish well between patients’ and control donors’ sera; only a small but statistically significant difference could be found between the mean IgG RAST value of the sera

lmmunoblot

technique

for IgE and IgG antibodies

541

m. Ek94 67 43 30 21.1 14.4

FIG. 4. lmmunoblot illustrating IgG antibodies to individual silk polypeptides in silk-sensitive patients (1 to 9) and in PCS (lane 10). The immunoblot was prepared as for Fig. 3 except that the second incubation was with ?-antihuman IgG.

from nine patients on one hand and that of the sera from 21 control donors on the other. More detailed information about the allergenic/antigenic activities of the SP were obtained by the immunoblot technique. The high resolving power of electrophoresis in polyacrylamide gels combined with the sensitivity of immunoblot made it possible to detect antibodies to the individual SP. The separation of SP carried out in this study was based mainly on differences in molecular weights making it possible to determine estimates of the molecular weights of the individual allergens. This information may serve as a starting point for the isolation of the individual allergenic polypeptides. The results of immunoblot analyses indicate that all electrophoretically resolvable polypeptides are capable of inducing IgE and IgG antibodies in susceptible persons, but not all susceptible persons are able to produce antibodies to all these polypeptides. This becomes especially clear when the spectrum of IgE antiSP antibodies are compared (Fig. 3). Serum No. 2 differed from all other sera in that it did not contain IgE antibodies to 40 kd polypeptides. Serum No. 5 contained no IgE antibodies to the minor polypeptides, whereas serum No. 4 appeared to contain antibodies to all resolvable SP. These differences are unlikely to be due to differences in specific IgE titers as measured

542

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by RAST, since sera Nos. 2, 4, and 5 elicited almost comparable IgE RAST values (Table II). According to the method of Sprague,‘” silk sericin consists of a group of polypeptides of molecular weights between 20 and 220 kd, whereas fibroin is composed mainly of two polypeptides of molecular weights about 350 kd. Based on molecular weight data, the allergenic polypeptides described in this article are more likely to belong to the sericin group of silk proteins; however, that some low molecular weight degradation products of fibroin are also involved cannot be excluded. We thank Ms. Barbara Jarosch for technical assistance and Ms. Comelia Fink for typing the manuscript. REFERENCES 1. Kino T, Oshima S: Allergy to insects in Japan. II. The reaginic sensitivity to silkworm moth in patients with bronchial asthma. J ALLERGYCLIN IMMLJNOL64:131, 1979 2. Kobayashi S: Occupational asthma due to inhalation of pharmacological dusts and other chemical agents with some reference to other occupational asthma in Japan. In Yamamura Y, Frick OL, Horiuchi Y, Kishimoto S, Miyamoto T, Naranjo P, DeWeck A, editors: Allergology. Proceedings of the VIII International Congress of Allergology. Amsterdam. 1974, Excerpta Medica. p 128 3. Fuchs E: Seide als allergen. Klinische untersuchung zur pathogenese von asthma bei seidenwebem. Dtsch Med Wochenschr 80:36, 1955

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4. Hacki M, Wihhrich B, Hauser M: Wildseide: ein aggressives inhalations allergen. Dtsch Med Wochenschr 107:166, 1982 5. Ziegler K: Seide. In Ullmann Enzyklopadie der technischen Chemie. 21:201, 1982, Verlag Chemie, GmbH 6. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat1 Acad Sci USA 76:4350. 1979 7. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265, 1951 8. Ceska M, Eriksson R, Varga JM: Radioimmunosorbent assay of allergens. J ALLERGYCLIN IMMUNOL 49: 1, 1972 9. Dewair M, Baur X: Radioallergosorbent test (RAST) for measurement of IgG antibodies to Aspergillusfumigatus in sera of patients with different lung diseases. J Immunol Methods 75:117, 1984 10. Shimizu M, Wither K, Reisman RE, Arbesman CE: Measurement of IgG antibodies to bee venom by radioallergosorbent test (RAST,). J ALLERGYCLIN IMMLJNOL57:211, 1976 11. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970 12. Batteiger B, Newhall WJ, Jones RB: The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods 55:297, 1981 13. Tsang VCW, Peratta JM, Simons AR: Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol 92:E377, 1983 14. Sprague KU: The Bombyx mori silk proteins: characterization of large polypeptides. Biochemistry 14:925, 1975