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Heterogeneity of grass pollen allergens (Dactylis glomerata) recognized by IgE antibodies in human patients sera by a new nitrocellulose immunoprint technique
Immunology Letters, 5 (1982) 127-131 Elsevier Biomedical Press
HETEROGENEITY OF GRASS POLLEN ALLERGENS (Dactylis glomerata) RECOGNIZED BY lgE ANTIBODIES IN HUMAN PATIENTS SERA BY A NEW NITROCELLULOSE IMMUNOPRINT TECHNIQUE Gabriel PELTRE, Josette LAPEYRE and Bernard DAVID Unit~ d'Immuno-Allergie, Institut Pasteur, 75015 Paris, France (Received 4 March 1982) (Modified version received and accepted 30 June 1982)
1. Summary A new nitrocellulose immunoprint technique has been developed to detect specific antigens or/and allergens present among a heterogeneous solution such as a water-soluble crude extract of a grass pollen (Daco'lis glomerata). The antigens are separated by isoelectric focusing (IEF) in an agarose gel and characterized by their isoelectric point (pl). These antigens a~e transferred and immobilized on a nitrocellulose sheet. They are recognized by the binding of specific antibodies contained in an unfractionated serum to be studied. Finally, the binding of these antibodies is visualized by species- or/and class-specific antibodies themselves labeled by an enzyme or by radioactivity. So one can detect the allergens recognized by the specific serum lgE antibodies and also the other antigens recognized by specific IgG, IgA or IgM antibodies.
2. Introduction The fact that lgE antibodies specific to allergens are mainly responsible of the allergic manifestations in immediate-type hypersensitivity is well known. Several immunochemical techniques are now available to detect and quantify IgE-specific antibodies, based upon radioimmunoassays (RIA such as the Radio-
Key words: allergens - human - IgE - nitrocellulose immunoprint
Allergo-So,bent Test or RAST) [ 1 I or enzymoinununoassays 12,3 ]. Other techniques were developed to study the aller gen or/and antigen heterogeneity in complex crude extracts responsible for immediate-lype hypersensitivity in humans. Among them the Crossed-Radiohnmuno-Electrophoresis (or CRIE) [4] is based upon the antigenic recognition by an animal serum of the potential allergens or antigens for humans. Another one, the qualitative allergen assay [5], uses an i,nmuno detection on a cyanogen bromide-activated cellulose print after an allergen extract isoelectric focusing (IEF) on polyacrylamide gel. In this work, we present the results obtained by using our new technique, the nitrocellulose immunoprint, to study the specificity of the lgE antibodies to a pollen extract of Dactylis glomerata, in human sera. We then compare the spectrotypes (or heterogeneity revealed by IEF) of the pollen extract recognized by IgE and lgG, lgA, lgM in an allergic patient serum and a control serum. Our technique is easy to perform and done in less than 24 h if enzymatic staining is used.
3. Materials and methods
3.1. Agarose isoelectric focusing The agarose IEF is performed on a fiat plastic sheet of Gelbond 1 I × 11 cm (Marine Colloids, Rockland, U.S.A.). The gel, 1 mm thick, consists of 1% agarose IEF (Pharmacia, Uppsala, Sweden), 12% sorbitol, 2%
0165-2478/82/0000-0000/$2.75 © 1982 Elsevier Biomedical Press
127
I'harmalytes, pH 3 - 1 0 (Pharmacia) or 2¢~ Servalytes, pll 4. 9 (Smwa, Ileidelber G, F.R.(,.). Tile crude extract of Dactylis" glomerata pollen is made just before use by suspending 50 mg of pollen Grains in 0.5 ml distilled water under agitation for 1 h at room temperature and then centrifuged 50 rain at 12,000g 200/.d of the supernatant is applied on a cellulose paper strip (0.5 × 10 cm) on the agarose gel at 3 cm from the cathode, parallel to the electrodes. 1EF is performed at 4°C at a conslant power of 6 W for q0 rain. The IEF equipment used is the FBE-3000 from Pharmacic, or the Multiphor from LKB. The constant power supply apparatus was either the I:'CPS 3000/150 from Pharmacia or the 2103 power supply fronl LKB. After focusilag an agarose strip, 2 cm wide, is cut off and stained by Coomassie blue as a control of the protein heterogeneity.
3.2. NitrocellldoSe print A print of the remaining agarose gel is taken on a nitrocelhflose sheet by blotting and pressing. This sheet, 10 × 11 cm (BA 85, 0.45 ~tm, Schleicher and Schfill, Dassel, F.R.G.), washed in distilled water, is applied on the agarosc gel, covered by a pad of absorbing paper, a glass plate and finally a 1 k G weirit for 5 rain. The wet absorbing paper is replaced and the gel is pressed again under 5 kg weight for 5 rain. All the absorbing paper is removed and any remaining moistt, re in the gel is removed through the nitrocellulose print by drying the Gel and the nitrocellulose sheet adhering to it under a fan for 10 -30 rain at room temperature.
3.3. Immtmochenlical detection The nitrocellulose print is then saturated in a 3% solution of bovine serum albumin (BSA) in phosphatebuffered saline (PBS) for I h at 45°C. The nitrocellulose print is then cut into 16 strips, each being 0.5 cm wide. Each strip is then incubated overnight at room temperature with 50- 200,ul of a pollen-sensitive patient's serum to be studied, diluted up to 600/.tl in PBS containing 0.1% Tween 20. The strip is rolled on the inner wall of a Petri dish (4 cm in diameter), the wall being perpendicular to the bottom of this dish, fitted with an air-tigllt cover (PVC culture dishes, reference 4917, Technofix, Paris, France). Such a closed chamber is fixed on a rotating drum (6 rpm) slightly tilted from the vertical position so that the 128
entire narrow nitrocellulose strip is kept in contact at each rew~lution of the drum, without dryinG, with the 600 ~1 of the patient's seruln dilution. The nitrocellulose strips are washed 6 times in 2 h in saline containing 0.1% Tween 20 and then incubated as previously with pure, labeled antibodies. Tilese antibodies are either anti-lgE, radioactively labeled with 12Sl (Pharlnacia RAST kit) diluted in PBS containing 104 Tween 20 to obtain 20,000-40,000 cpm in a final volume of 600 ~1/ 0.5 cm wide ,litrocellulose strip, or anti-lgG, -lgA or -IgM labeled with horseradish peroxidase, obtained froln Institut Pasteur Production (Paris, France) diluted 300 times (600 ,ul/0.5 cm wide nitrocellulose strip). These antibodies are incubated for 2 h at room temperature. After washing 6 times in 2 h in saline containing 0.1% Tween 20, the nitrocellt, lose strips to be autoradiographed arc dried and exposed t, nder close contact with a Kodak-X-Omat film. Tire strips incubated with enzyme-labeled antiserum arc incubated with the Dialnino Benzidine stain for peroxydase.
3.4. Radioimmuno assays Total lgE were detected by PRIST-Phadebas [61 and expressed in ng lgE/ml sert, m. Specific lgE antibodies to Dact)'lis glomerata pollen were detected by RAST-Phadebas [ 1] on undiluted sera. The results were expressed as the ratio between the radioactivity fixed to immobilized pollen extract by the patient's serum compared to the radioactivity bound by a pool of sera l'ronl non sensitive donors.
4. Results
4. I. Hett'rogeneio' eft"the allergens recognized b.v serum IgE antibodies from Dactylis glomeratasensitive patien ts In Fig. 1. the heterogeneity of the l)actylis glomerata pollen crude extract visualized by Coomassie blue staining reveals at least 60 bands, their pls ranging from 3.9-10.5. Such a pattern is called an IEF spectrotype. Tile nitrocellulose print of this spectrotype was cut into 16 narrow strips and each strip was processed as described in Materials and Methods. Each strip represents the heterogeneity of the pollen fractions to which serum lgE from one single patient is bound, that is by definition the allergen fractions
Table 1
pH 4-
6_ m ~ O
7-
m
tim
I
I04-
,,,,.
i(~
,~
14
!a
*;t
I~
iO
9
~
'
,5
~
a
3
)
Patient's no.
Total IgE (ng/ml)
Specific Igt-a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
5600 40 120 3200 370 10 800 2200 not done 30 370 1520 370 1240 1240 850 300
34 10 12 18 20 30 30 20 14 25 31 28 33 34 33 28
1
Fig. 1. lteterogeneity of the allergens recognized by the serum IgE from 16 patients sensitive to the pollen extract. The IEF band on the left shows the heterogeneity of the whole pollen extract "after IEF, stained by Coomassie blue. The allergens are reveaied by autoradiography.
recognized by each patient's lgE. A great difference can be seen front one patient to the other among the 16 cases shown in Fig. 1. Some serum lgE such as those from patients no. 3, 6, 9 and 12 detect a very restricted spectrotype: the same single band is detected. Patients 2 and 7 have IgE recognizing a few more acidic IEF bands, whereas patients 11 and 16 have IgE binding a still more basic IEF bands. Patient 8 has IgE antibodies binding more than 13 allergens bands. Finally the sera of patients 1, 10, 13, 1 4 a n d 15 are able to recognize even more pollen IEF bands, more than 30 bands. So all the patients studied do recognize at least one common band at pl 5.9, the most frequent allergen for them and often the most intensely labeled IEF band. One exception, however, is seen with the serum no. 5 where this common band is weaker than with the others. One can also see that several IEF bands recognized by a patient's lgE antibodies are not stained by the Coomassie staining, the reverse also being true. Table 1 shows the total lgE amounts in ng/ml serum and the levels o f specific IgE to Dactylis pollen as compared to a pool o f sera from non-sensitive
aExpressed as the ratio between the radioactivity fixed to immobilized pollen extract by the patients' serum compared to the radioactivity bound by a pool of sera from non-sensitive donors. donors (see Materials and methods section). Front our own experience the mean total seric lgE level in norreal individuals is lower than 300 ng/ml. Four patients out of the 15 we have shown to have a normal level of total seric lgE. There is not obvious relationship between the specific IgE level to Daco'lis pollen extract and the total IgE level in the patients studied. Their is a rather good agreement between the RAST values and the intensity of the radioactive labeling o f the nitrocellulose immunoprints if one compares spectrotypes of equal heterogeneity. However, patient 6, who recognizes only one allergen in the crude extract of Dactylis pollen, has far more lgE antibodies (RAST value: 30) to this single IEF band titan patient 8 (RAST value: 20) who recognizes several allergens in the same pollen extract. This is also true for patients 10 and 12. These differences in quantity of specific IgE are, however, suggested by the intensity of the ",allergen bands revealed by autoradiography. 4.2. Specific antibodies to pollen extracts from a normal individual as compared to those from a pollen-sensitive pa tien t In Fig. 2 are shown from left to right the IEF spectrotypes of tile pollen antigens recognized by the IgG, IgA, IgM and IgE from a normal individual. The first 129
pH -4
...~.. .
.
.
.
-9
Ig
G
A
M
Normlil serum
I E i I
E
G
A
M
Coo
Patjenl serum
l ig. 2. II-I' spectrotypes of the pollen antigens and allergens recognized by the IgG, IgA, IgM and IgE from a normal serum and from a patienls's serum.
3 nitrocellulose inununoprints were developed using horseradisli peroxidase-labeled :.illti-lllllllUUOglobuJin class antibodies, ]'he last strip represents an autoradiogram obtained after 3 weeks of exposure, the anti-lg[ antibodies used being radioactively labeled as mentioned in the Materials and Methods section. No IEl" band is visible. The next 4 bands represent the allergens and antigens recognized, respectively, by the pollen-sensitive patient's IgE, lgG, IgA and lgM. The last baud is the pollen extract stained by Coomassie blue. This patient is obviously sensitive to a great number of allergens as can be seen on the strip labeled E. One can observe that nearly all the pollen IEF bands recognized by the patient's lgE antibodies are also recognized by the other classes of antibodies, ltowever, the lgG, IgA and IgM antibodies are recognizing more bands than the lgL: antibodies particularly in the acidic p l I range. 5. Discussion
Based upon IEF as the electrophoretic techniqt, e to separate the allergen extract components, as done 130
earlier by Ceska [6] and Brighton [7i, our technique profits from tile great advantages of this separation: high resolution of a complex extract in sharply focused bands and characterization of these bands by their isoelectric points. However, tile IEF separation presents some limitations. Sometimes it reveals a physicochemical microheterogeneity in some proteins with no or yet t, nrecognized physiological relevance. The fact that some patients" IgE recognize only one pollen band, as the one focused at ptl 5.9, suggests homogeneity and maybe the uniqueness of this band after IEF. The nitrocellulose sheet used to make the print of all electrophorelic separation, as introduced by Southern [81, binds ram-specifically the pollen extract components. Our easy and fast (30 rain) method to transfer the pollen components f1o111 the IEF agarose gel to the nitrocellulose sheet gives very sharp bands after immune-detection and leaves a very limited background staining. No protein bands detected b) Coomassie blue staining, remain on the agarose gel after their transfer onto the nitrocellulose print. Our technique is mainly qualitative as we do not know if the amoui/t of allergen bound to the nitrocellulose print is in such a large excess to allow a quantitative comparison between high and low specific lgE-ctmtaining sera. I lowever, due to the rather good relationship between the RAST values and the intensity of the labeling of the prints (Fig. 1) we consider this technique as semi-quantitative. Fig. 1 shows a great helerogeneity of the allergens detectable by some patients" serum igE. More than 30 IEF bands were recognized covering a wide pit range, from 4.1 10.5. Among the l6 patients analyzed iu Fig. I one can distinguish roughly 3 types of lgli responses to the pollen extract: a very restricted response, only one II'F band for 4 patients; a heterogeneous response for 8 patients; and a ve,y heterogeneous response for 4 patients. Could these spectretypes be the expression of different classes of phenotypes': As all our patients live m the same i~ei~lbot, ilined, we assume that thev were all exposed to the same pollinic stimulus. The fact that they exhibit such a diversified IgE response to the pollen components suggests either a genetic modulation of the allergic response or it reflects the degree of maturation of their disease. The patients recognizing only one pollen allergen may recognize other minor allergens due to the limit of sensitivity of the detection method.
This is particularly true for the sera with low RAST values(Fig. I and Table 1, patient 3). A true restricted heterogeneity of allergen recognition can, however, be found with high RAST values (Fig. 1 and Table 1, patients 6 and 1 2). In Fig. 2, we four_d that IgG, IgA and IgM antibodies were produced to a great variety of pollen components. Some striking differences were seen between the spectrotypes obtained with lgG, IgA or lgM from these two sera. Such a variation may have the same origin as the one suggested above for the lgE response. Fig. 2 shows also that nearly all the allergens recognized by lgE antibodies are also recognized by antibodies from the other immunoglobulin classes. As the patient studied really suffers from its pollen allergen it is obvious that the non-lgE antibodies are not blocking the effect of the IgE antibodies. Maybe the specificity of-the 'blocking antibodies' is different from the specificity of the IgE antibodies for the same allergen molecules: some epitopes are recognized by the IgE antibodies and others on the same molecule by the IgG, lgA or lgM antibodies. Maybe the amount or the affinity of the IgE antibodies to pollen is greater
than the amount or affinity of the other inuuunoglobulin classes to tile same allergens, in such a way that specific lgG, IgA and IgM cannot avoid the pollen components to reach the target cells (mast cells and basophils), cove,ed with lgE antibodies.
References [1 ] Wide, L., Bennich, 11. and Johansson, S. G. O. (1967) Lancet II, 1105. [21 Ericksson, N. E. and Ahlstedts, L. (1977) Int. Arch. Allergy Appl. lmmunol. 54, 88. [3] Guesdon, J. L., David, B. and Lapeyre, J. (1978) Clin. Exp. lmmunol. 33,430--436. [4] Weeke, B. and Lowenstein, I1. (1973) Stand. J. Immunol. 2, Suppl. 1, 149-153. [5] Schr6der, If., Blomberg, F. and Yman, L. (1980) in: Advances in Allergology and Clinical Immunology, (Oehling, A., Mafllov, E., Glazer, i. and Arbesman, G. Eds.), Pergamon Press, pp. 513-519. [6] Ceska, M. (1972) Int. Arch. Allergy 43,419-426. [7] Brighton, W. D. (1975) in: Developments in Biological Standardization, Vol. 29, S. Karger, Basel-Mfinchen-ParisLondon-New York-Sydney, pp. 362-369. [8] Southern, E. M. (1975) J. Mol. Biol. 98,503-517.
131
HETEROGENEITY OF GRASS POLLEN ALLERGENS (Dactylis glomerata) RECOGNIZED BY lgE ANTIBODIES IN HUMAN PATIENTS SERA BY A NEW NITROCELLULOSE IMMUNOPRINT TECHNIQUE Gabriel PELTRE, Josette LAPEYRE and Bernard DAVID Unit~ d'Immuno-Allergie, Institut Pasteur, 75015 Paris, France (Received 4 March 1982) (Modified version received and accepted 30 June 1982)
1. Summary A new nitrocellulose immunoprint technique has been developed to detect specific antigens or/and allergens present among a heterogeneous solution such as a water-soluble crude extract of a grass pollen (Daco'lis glomerata). The antigens are separated by isoelectric focusing (IEF) in an agarose gel and characterized by their isoelectric point (pl). These antigens a~e transferred and immobilized on a nitrocellulose sheet. They are recognized by the binding of specific antibodies contained in an unfractionated serum to be studied. Finally, the binding of these antibodies is visualized by species- or/and class-specific antibodies themselves labeled by an enzyme or by radioactivity. So one can detect the allergens recognized by the specific serum lgE antibodies and also the other antigens recognized by specific IgG, IgA or IgM antibodies.
2. Introduction The fact that lgE antibodies specific to allergens are mainly responsible of the allergic manifestations in immediate-type hypersensitivity is well known. Several immunochemical techniques are now available to detect and quantify IgE-specific antibodies, based upon radioimmunoassays (RIA such as the Radio-
Key words: allergens - human - IgE - nitrocellulose immunoprint
Allergo-So,bent Test or RAST) [ 1 I or enzymoinununoassays 12,3 ]. Other techniques were developed to study the aller gen or/and antigen heterogeneity in complex crude extracts responsible for immediate-lype hypersensitivity in humans. Among them the Crossed-Radiohnmuno-Electrophoresis (or CRIE) [4] is based upon the antigenic recognition by an animal serum of the potential allergens or antigens for humans. Another one, the qualitative allergen assay [5], uses an i,nmuno detection on a cyanogen bromide-activated cellulose print after an allergen extract isoelectric focusing (IEF) on polyacrylamide gel. In this work, we present the results obtained by using our new technique, the nitrocellulose immunoprint, to study the specificity of the lgE antibodies to a pollen extract of Dactylis glomerata, in human sera. We then compare the spectrotypes (or heterogeneity revealed by IEF) of the pollen extract recognized by IgE and lgG, lgA, lgM in an allergic patient serum and a control serum. Our technique is easy to perform and done in less than 24 h if enzymatic staining is used.
3. Materials and methods
3.1. Agarose isoelectric focusing The agarose IEF is performed on a fiat plastic sheet of Gelbond 1 I × 11 cm (Marine Colloids, Rockland, U.S.A.). The gel, 1 mm thick, consists of 1% agarose IEF (Pharmacia, Uppsala, Sweden), 12% sorbitol, 2%
0165-2478/82/0000-0000/$2.75 © 1982 Elsevier Biomedical Press
127
I'harmalytes, pH 3 - 1 0 (Pharmacia) or 2¢~ Servalytes, pll 4. 9 (Smwa, Ileidelber G, F.R.(,.). Tile crude extract of Dactylis" glomerata pollen is made just before use by suspending 50 mg of pollen Grains in 0.5 ml distilled water under agitation for 1 h at room temperature and then centrifuged 50 rain at 12,000g 200/.d of the supernatant is applied on a cellulose paper strip (0.5 × 10 cm) on the agarose gel at 3 cm from the cathode, parallel to the electrodes. 1EF is performed at 4°C at a conslant power of 6 W for q0 rain. The IEF equipment used is the FBE-3000 from Pharmacic, or the Multiphor from LKB. The constant power supply apparatus was either the I:'CPS 3000/150 from Pharmacia or the 2103 power supply fronl LKB. After focusilag an agarose strip, 2 cm wide, is cut off and stained by Coomassie blue as a control of the protein heterogeneity.
3.2. NitrocellldoSe print A print of the remaining agarose gel is taken on a nitrocelhflose sheet by blotting and pressing. This sheet, 10 × 11 cm (BA 85, 0.45 ~tm, Schleicher and Schfill, Dassel, F.R.G.), washed in distilled water, is applied on the agarosc gel, covered by a pad of absorbing paper, a glass plate and finally a 1 k G weirit for 5 rain. The wet absorbing paper is replaced and the gel is pressed again under 5 kg weight for 5 rain. All the absorbing paper is removed and any remaining moistt, re in the gel is removed through the nitrocellulose print by drying the Gel and the nitrocellulose sheet adhering to it under a fan for 10 -30 rain at room temperature.
3.3. Immtmochenlical detection The nitrocellulose print is then saturated in a 3% solution of bovine serum albumin (BSA) in phosphatebuffered saline (PBS) for I h at 45°C. The nitrocellulose print is then cut into 16 strips, each being 0.5 cm wide. Each strip is then incubated overnight at room temperature with 50- 200,ul of a pollen-sensitive patient's serum to be studied, diluted up to 600/.tl in PBS containing 0.1% Tween 20. The strip is rolled on the inner wall of a Petri dish (4 cm in diameter), the wall being perpendicular to the bottom of this dish, fitted with an air-tigllt cover (PVC culture dishes, reference 4917, Technofix, Paris, France). Such a closed chamber is fixed on a rotating drum (6 rpm) slightly tilted from the vertical position so that the 128
entire narrow nitrocellulose strip is kept in contact at each rew~lution of the drum, without dryinG, with the 600 ~1 of the patient's seruln dilution. The nitrocellulose strips are washed 6 times in 2 h in saline containing 0.1% Tween 20 and then incubated as previously with pure, labeled antibodies. Tilese antibodies are either anti-lgE, radioactively labeled with 12Sl (Pharlnacia RAST kit) diluted in PBS containing 104 Tween 20 to obtain 20,000-40,000 cpm in a final volume of 600 ~1/ 0.5 cm wide ,litrocellulose strip, or anti-lgG, -lgA or -IgM labeled with horseradish peroxidase, obtained froln Institut Pasteur Production (Paris, France) diluted 300 times (600 ,ul/0.5 cm wide nitrocellulose strip). These antibodies are incubated for 2 h at room temperature. After washing 6 times in 2 h in saline containing 0.1% Tween 20, the nitrocellt, lose strips to be autoradiographed arc dried and exposed t, nder close contact with a Kodak-X-Omat film. Tire strips incubated with enzyme-labeled antiserum arc incubated with the Dialnino Benzidine stain for peroxydase.
3.4. Radioimmuno assays Total lgE were detected by PRIST-Phadebas [61 and expressed in ng lgE/ml sert, m. Specific lgE antibodies to Dact)'lis glomerata pollen were detected by RAST-Phadebas [ 1] on undiluted sera. The results were expressed as the ratio between the radioactivity fixed to immobilized pollen extract by the patient's serum compared to the radioactivity bound by a pool of sera l'ronl non sensitive donors.
4. Results
4. I. Hett'rogeneio' eft"the allergens recognized b.v serum IgE antibodies from Dactylis glomeratasensitive patien ts In Fig. 1. the heterogeneity of the l)actylis glomerata pollen crude extract visualized by Coomassie blue staining reveals at least 60 bands, their pls ranging from 3.9-10.5. Such a pattern is called an IEF spectrotype. Tile nitrocellulose print of this spectrotype was cut into 16 narrow strips and each strip was processed as described in Materials and Methods. Each strip represents the heterogeneity of the pollen fractions to which serum lgE from one single patient is bound, that is by definition the allergen fractions
Table 1
pH 4-
6_ m ~ O
7-
m
tim
I
I04-
,,,,.
i(~
,~
14
!a
*;t
I~
iO
9
~
'
,5
~
a
3
)
Patient's no.
Total IgE (ng/ml)
Specific Igt-a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
5600 40 120 3200 370 10 800 2200 not done 30 370 1520 370 1240 1240 850 300
34 10 12 18 20 30 30 20 14 25 31 28 33 34 33 28
1
Fig. 1. lteterogeneity of the allergens recognized by the serum IgE from 16 patients sensitive to the pollen extract. The IEF band on the left shows the heterogeneity of the whole pollen extract "after IEF, stained by Coomassie blue. The allergens are reveaied by autoradiography.
recognized by each patient's lgE. A great difference can be seen front one patient to the other among the 16 cases shown in Fig. 1. Some serum lgE such as those from patients no. 3, 6, 9 and 12 detect a very restricted spectrotype: the same single band is detected. Patients 2 and 7 have IgE recognizing a few more acidic IEF bands, whereas patients 11 and 16 have IgE binding a still more basic IEF bands. Patient 8 has IgE antibodies binding more than 13 allergens bands. Finally the sera of patients 1, 10, 13, 1 4 a n d 15 are able to recognize even more pollen IEF bands, more than 30 bands. So all the patients studied do recognize at least one common band at pl 5.9, the most frequent allergen for them and often the most intensely labeled IEF band. One exception, however, is seen with the serum no. 5 where this common band is weaker than with the others. One can also see that several IEF bands recognized by a patient's lgE antibodies are not stained by the Coomassie staining, the reverse also being true. Table 1 shows the total lgE amounts in ng/ml serum and the levels o f specific IgE to Dactylis pollen as compared to a pool o f sera from non-sensitive
aExpressed as the ratio between the radioactivity fixed to immobilized pollen extract by the patients' serum compared to the radioactivity bound by a pool of sera from non-sensitive donors. donors (see Materials and methods section). Front our own experience the mean total seric lgE level in norreal individuals is lower than 300 ng/ml. Four patients out of the 15 we have shown to have a normal level of total seric lgE. There is not obvious relationship between the specific IgE level to Daco'lis pollen extract and the total IgE level in the patients studied. Their is a rather good agreement between the RAST values and the intensity of the radioactive labeling o f the nitrocellulose immunoprints if one compares spectrotypes of equal heterogeneity. However, patient 6, who recognizes only one allergen in the crude extract of Dactylis pollen, has far more lgE antibodies (RAST value: 30) to this single IEF band titan patient 8 (RAST value: 20) who recognizes several allergens in the same pollen extract. This is also true for patients 10 and 12. These differences in quantity of specific IgE are, however, suggested by the intensity of the ",allergen bands revealed by autoradiography. 4.2. Specific antibodies to pollen extracts from a normal individual as compared to those from a pollen-sensitive pa tien t In Fig. 2 are shown from left to right the IEF spectrotypes of tile pollen antigens recognized by the IgG, IgA, IgM and IgE from a normal individual. The first 129
pH -4
...~.. .
.
.
.
-9
Ig
G
A
M
Normlil serum
I E i I
E
G
A
M
Coo
Patjenl serum
l ig. 2. II-I' spectrotypes of the pollen antigens and allergens recognized by the IgG, IgA, IgM and IgE from a normal serum and from a patienls's serum.
3 nitrocellulose inununoprints were developed using horseradisli peroxidase-labeled :.illti-lllllllUUOglobuJin class antibodies, ]'he last strip represents an autoradiogram obtained after 3 weeks of exposure, the anti-lg[ antibodies used being radioactively labeled as mentioned in the Materials and Methods section. No IEl" band is visible. The next 4 bands represent the allergens and antigens recognized, respectively, by the pollen-sensitive patient's IgE, lgG, IgA and lgM. The last baud is the pollen extract stained by Coomassie blue. This patient is obviously sensitive to a great number of allergens as can be seen on the strip labeled E. One can observe that nearly all the pollen IEF bands recognized by the patient's lgE antibodies are also recognized by the other classes of antibodies, ltowever, the lgG, IgA and IgM antibodies are recognizing more bands than the lgL: antibodies particularly in the acidic p l I range. 5. Discussion
Based upon IEF as the electrophoretic techniqt, e to separate the allergen extract components, as done 130
earlier by Ceska [6] and Brighton [7i, our technique profits from tile great advantages of this separation: high resolution of a complex extract in sharply focused bands and characterization of these bands by their isoelectric points. However, tile IEF separation presents some limitations. Sometimes it reveals a physicochemical microheterogeneity in some proteins with no or yet t, nrecognized physiological relevance. The fact that some patients" IgE recognize only one pollen band, as the one focused at ptl 5.9, suggests homogeneity and maybe the uniqueness of this band after IEF. The nitrocellulose sheet used to make the print of all electrophorelic separation, as introduced by Southern [81, binds ram-specifically the pollen extract components. Our easy and fast (30 rain) method to transfer the pollen components f1o111 the IEF agarose gel to the nitrocellulose sheet gives very sharp bands after immune-detection and leaves a very limited background staining. No protein bands detected b) Coomassie blue staining, remain on the agarose gel after their transfer onto the nitrocellulose print. Our technique is mainly qualitative as we do not know if the amoui/t of allergen bound to the nitrocellulose print is in such a large excess to allow a quantitative comparison between high and low specific lgE-ctmtaining sera. I lowever, due to the rather good relationship between the RAST values and the intensity of the labeling of the prints (Fig. 1) we consider this technique as semi-quantitative. Fig. 1 shows a great helerogeneity of the allergens detectable by some patients" serum igE. More than 30 IEF bands were recognized covering a wide pit range, from 4.1 10.5. Among the l6 patients analyzed iu Fig. I one can distinguish roughly 3 types of lgli responses to the pollen extract: a very restricted response, only one II'F band for 4 patients; a heterogeneous response for 8 patients; and a ve,y heterogeneous response for 4 patients. Could these spectretypes be the expression of different classes of phenotypes': As all our patients live m the same i~ei~lbot, ilined, we assume that thev were all exposed to the same pollinic stimulus. The fact that they exhibit such a diversified IgE response to the pollen components suggests either a genetic modulation of the allergic response or it reflects the degree of maturation of their disease. The patients recognizing only one pollen allergen may recognize other minor allergens due to the limit of sensitivity of the detection method.
This is particularly true for the sera with low RAST values(Fig. I and Table 1, patient 3). A true restricted heterogeneity of allergen recognition can, however, be found with high RAST values (Fig. 1 and Table 1, patients 6 and 1 2). In Fig. 2, we four_d that IgG, IgA and IgM antibodies were produced to a great variety of pollen components. Some striking differences were seen between the spectrotypes obtained with lgG, IgA or lgM from these two sera. Such a variation may have the same origin as the one suggested above for the lgE response. Fig. 2 shows also that nearly all the allergens recognized by lgE antibodies are also recognized by antibodies from the other immunoglobulin classes. As the patient studied really suffers from its pollen allergen it is obvious that the non-lgE antibodies are not blocking the effect of the IgE antibodies. Maybe the specificity of-the 'blocking antibodies' is different from the specificity of the IgE antibodies for the same allergen molecules: some epitopes are recognized by the IgE antibodies and others on the same molecule by the IgG, lgA or lgM antibodies. Maybe the amount or the affinity of the IgE antibodies to pollen is greater
than the amount or affinity of the other inuuunoglobulin classes to tile same allergens, in such a way that specific lgG, IgA and IgM cannot avoid the pollen components to reach the target cells (mast cells and basophils), cove,ed with lgE antibodies.
References [1 ] Wide, L., Bennich, 11. and Johansson, S. G. O. (1967) Lancet II, 1105. [21 Ericksson, N. E. and Ahlstedts, L. (1977) Int. Arch. Allergy Appl. lmmunol. 54, 88. [3] Guesdon, J. L., David, B. and Lapeyre, J. (1978) Clin. Exp. lmmunol. 33,430--436. [4] Weeke, B. and Lowenstein, I1. (1973) Stand. J. Immunol. 2, Suppl. 1, 149-153. [5] Schr6der, If., Blomberg, F. and Yman, L. (1980) in: Advances in Allergology and Clinical Immunology, (Oehling, A., Mafllov, E., Glazer, i. and Arbesman, G. Eds.), Pergamon Press, pp. 513-519. [6] Ceska, M. (1972) Int. Arch. Allergy 43,419-426. [7] Brighton, W. D. (1975) in: Developments in Biological Standardization, Vol. 29, S. Karger, Basel-Mfinchen-ParisLondon-New York-Sydney, pp. 362-369. [8] Southern, E. M. (1975) J. Mol. Biol. 98,503-517.
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