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Purification of allergens by high-performance liquid chromatography
Journal of Chromatography,
408 (I 987) 420-425
Elsevier Science Publishers B.V., Amsterdam -
Printed in The Netherlands
CHROM. I9 847
Note
Purification
of allergens by high-performance
WI. Purification
liquid chromatography
of the major allergen of birch pollen (Betuh
werru-
COS8)
NAZARIO RUBIO* Institute Cajal, Velazquez 144, 28006 Madrid (Spain)
and AURORA BRIEVA Luboratorios Andromaco, S.A., Allergy Division. Torrejon de Ardoz (Spain)
(Received July 6th, 1987)
Birch pollen is one of the main sources of allergens in countries in northern Europa. Several attempts of purificaton and immunochemicai characterization of the major allergen from this species have been made1-4. As the last note of this series dealing with the purification of allergens5-10, we report the rapid purification of the major allergen, of Bet& verrucosa pollen. EXPERIMENTAL
Allergen extraction
Pollen from Be&la verrucosa was obtained from Allergon (Angelholm, Sweden) and contain 1% non-pollen debris. It was extracted as described in ref. 5. High-performance
liquid chromatography
(HPLC)
HLC was performed as described in previous papers of this series5. Immunological methods
Solid-phase radioimmunoassay (SPRIA) and the radioallergosorbent (RAST) were carried out according to refs. 5 and 6.
test
Polyacrylamide slab gel electrophoresis
The method described by Laemmli” was employed. The separating gel consisted of 12.5% polyacrylamide and the stacking gel contained 4.5% of polyacrylamide. Gels were stained in 0.25% Coomassie Brilliant Blue G-250 prepared in methanol-acetic acid-water (45: 10:45), for 1 h. RESULTS
PuriJicatiun of the allergen
The position of the major allergen of B. verrucosa in the HPLC chromatogram 0021-9673/87/$03.50
0
1987 Elsevier Science Publishers B.V.
NOTES
421
was determined by performing solid-phase RIA of each fraction (Fig. 1). With the three high-activity sera employed, a clear distribution of the activity in fractions surrounding fraction number 10 was detected (hatched area). No allergenic activity was found in the low-molecular-weight material eluted from the column. When fraction 10 was rechromatographed at higher sensitivity, a single peak with a retention time of 10.55 min was found (Fig. 2). The data recorded indicate 90% purity. Fraction 11 from this rechromatography contains the allergen almost 100% pure. Molecular weight detemination gave a value of 17 000 daltons, in accordance with previously reported data4. Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Fig. 3 shows the electrophoresis
FRACTION
(SDS-PAGE)
of fraction 10 from the f&t separation.
The
NUMBER
Fig. I. HPLC separation of Bet& verrucosa pollen extract. A Protein Pack 125 column and phosphatebuffered saline (PBS) as eluent was used. Three different allergic sera were tested in SPRIA against the separated fractions (histograms).
422
NOTES
r RT= iO.51
RETENTION
Fig. 2. Rechromatography
TIME
(MINUTES)
of HPLC fraction 10 from Fig. 1. RT = Retention time in min.
Fig. 3. SDS-PAGE of birch main allergen purified by HPLC. Lanes: A = molecular weight markers, phosphorylase 6 (mol. wt. 94000), bovine serum albumin (67 000), ovalbmnin (43 000), carbonic anhydrase (30000). soybean trypsin inhibitor (20 100) and cr-lactalbumin (14000): B = purified allergen.
NOTES
423
kg
OF ALLERGEN
Fig. 4. Titration of birch crude extract (m) and HPLC-purified main allergen (0). A pool of allergic sera (class 4 RAST) and 1251-labelled rabit anti-human IgE was used in SPRIA.
.-
pure allergen (lane B) showed a molecular weight value of 17 000 daltons, in agreement with the value detemined by HPLC. Immunological properties
To characterize further the purified allergen from an immunological view, we compared it with the crude extract in SPRIA.
point of
loo-
m l
a /
g
E . I af
* /
so-
. ./
l
-I
I
loo0
I 1W
I
10
I 1
RECIPROCAL OF ALLERGEN DILUTION
Fig. 5. RAST inhibition assay. The results are expressed as the percentage inhibition of binding of specific IgE achieved by addition of serial dilutions of allergen. The undiluted Pharmalgens extract corresponds to 100000 B.U./ml. n = HPLC-purified allergen: l = Pharmalgena extract.
424
NOTES
A dose-dependent binding of specific immunoglobin E (IgE), as revealed by the amount of r2+labelled rabbit antibody fixed to the wells, was obtained in both cases (Fig. 4). To reach 50% binding of radiactivity, 0.2 ,ng of the pure allergen and 1 pg of crude extract, are needed. Thus, the final specific activity of the purified protein is about five-fold greater than that of the starting material, in accord with the contribution of the allergen peak to the total amount of proteins (Fig. 1). The inhibition of RAST gave identical results to those obtained with the Pharmalgenm purified extract (Fig. 5). The same pool of sera from the patients used in Fig. 4 was used as a source of specific anti Betula IgE. 1000 Biological Units (B.U.) were required to achieve 50% inhibition in R&T, which corresponds to 1.6 pg of pure protein, as determined by the method of Lowry et a1.12. Hence, 1 B.U. is equivalent to 1.6 ng of pure B. verrucosa allergen. DISCUSSION
In this, the final communication of our recent work dealing with the purification of allergens from vegetal, fungal, mites or mammalian origin, we present the HPLC technology needed for a rapid and quantitative purification of the major allergen of birch pollen. As described previously J- lo, HPLC Protein Pack 125 columns and phosphate-buffered saline allow rapid separations of proteins in crude extracts as a function of their molecular weights. A specific immunoassay with sera from atopic patients provides us with a method for the localization of allergens in the fractions obtained after HPLC separation. The Be&la verrucosa main allergen is eiuted from the column in almost pure form in fraction 10 (Fig. 2). Its purity was checked by SDS-PAGE (Fig. 3) and its molecular weight, determined by HPLC and PAGE, corresponds with the values previously reported by other authors. The pure allergen, as is a rule in HPLC purification, retains its full immunological properties of IgE binding. This was demonstrated by titration against specific IgE-containing human sera (Fig. 4) and RAST inhibition (Fig. 5). The purified mollecule is enriched about. five-fold in comparison with a crude birch pollen extract, and we also determined that 1.6 ng are needed to produce 1 B.U. The industrial-scale purification of the allergen can provide allergic patients with a specific treatment instead of the semicrude-extract therapy currently used over the world. ACKNOWLEDGEMENT
The technical assistance of Miss Mercedes Alonso is gratefully acknowledged. REFERENCES 1 L. Belin, ht. Arch. Allergy Appl. Immunol., 42 (1972) 300. 2 M. Viander, J. Fraki, B. M. Djupsund and S. Laine, AI1erg.y.34 (1979) 289.
NOTES 3 4 5 6 7 8 9 10 11 12
J. Apold, E. Florvaag and S. Elsayed, Int. Arch. ANergy Appl. Immunol., 64 (1981) 439. H. Ipsen and H. Lowenstein, J. Allergy Clin. Zmmunol., 72 (1983) 150. A. Brieva and N. Rubio, J. Chromutogr., 370 (1986) 165. N. Rubio, A. Brieva and M. Rubio, J. Chromatogr., 392 (1987) 447. N. Rubio, A. Brieva and B. Alcazar, J. Chromatogr., 403 (1987) 312. N. Rubio and A. Brieva, J. Chromatogr., 404 (1987) 378. N. Rubio and A. Brieva, J. Chromatogr., 398 (1987) 366. N. Rubio and A. Brieva, J. Chromatogr., 407 (1987) 408. U. K. Laemmli, Nature (London), 227 (1970) 680. 0. H. Lowry, N. J. Rosebrough, A. L. Fan and R. L Randall, J. Biol. Chem., I93 (1951)
42.5
265.
408 (I 987) 420-425
Elsevier Science Publishers B.V., Amsterdam -
Printed in The Netherlands
CHROM. I9 847
Note
Purification
of allergens by high-performance
WI. Purification
liquid chromatography
of the major allergen of birch pollen (Betuh
werru-
COS8)
NAZARIO RUBIO* Institute Cajal, Velazquez 144, 28006 Madrid (Spain)
and AURORA BRIEVA Luboratorios Andromaco, S.A., Allergy Division. Torrejon de Ardoz (Spain)
(Received July 6th, 1987)
Birch pollen is one of the main sources of allergens in countries in northern Europa. Several attempts of purificaton and immunochemicai characterization of the major allergen from this species have been made1-4. As the last note of this series dealing with the purification of allergens5-10, we report the rapid purification of the major allergen, of Bet& verrucosa pollen. EXPERIMENTAL
Allergen extraction
Pollen from Be&la verrucosa was obtained from Allergon (Angelholm, Sweden) and contain 1% non-pollen debris. It was extracted as described in ref. 5. High-performance
liquid chromatography
(HPLC)
HLC was performed as described in previous papers of this series5. Immunological methods
Solid-phase radioimmunoassay (SPRIA) and the radioallergosorbent (RAST) were carried out according to refs. 5 and 6.
test
Polyacrylamide slab gel electrophoresis
The method described by Laemmli” was employed. The separating gel consisted of 12.5% polyacrylamide and the stacking gel contained 4.5% of polyacrylamide. Gels were stained in 0.25% Coomassie Brilliant Blue G-250 prepared in methanol-acetic acid-water (45: 10:45), for 1 h. RESULTS
PuriJicatiun of the allergen
The position of the major allergen of B. verrucosa in the HPLC chromatogram 0021-9673/87/$03.50
0
1987 Elsevier Science Publishers B.V.
NOTES
421
was determined by performing solid-phase RIA of each fraction (Fig. 1). With the three high-activity sera employed, a clear distribution of the activity in fractions surrounding fraction number 10 was detected (hatched area). No allergenic activity was found in the low-molecular-weight material eluted from the column. When fraction 10 was rechromatographed at higher sensitivity, a single peak with a retention time of 10.55 min was found (Fig. 2). The data recorded indicate 90% purity. Fraction 11 from this rechromatography contains the allergen almost 100% pure. Molecular weight detemination gave a value of 17 000 daltons, in accordance with previously reported data4. Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Fig. 3 shows the electrophoresis
FRACTION
(SDS-PAGE)
of fraction 10 from the f&t separation.
The
NUMBER
Fig. I. HPLC separation of Bet& verrucosa pollen extract. A Protein Pack 125 column and phosphatebuffered saline (PBS) as eluent was used. Three different allergic sera were tested in SPRIA against the separated fractions (histograms).
422
NOTES
r RT= iO.51
RETENTION
Fig. 2. Rechromatography
TIME
(MINUTES)
of HPLC fraction 10 from Fig. 1. RT = Retention time in min.
Fig. 3. SDS-PAGE of birch main allergen purified by HPLC. Lanes: A = molecular weight markers, phosphorylase 6 (mol. wt. 94000), bovine serum albumin (67 000), ovalbmnin (43 000), carbonic anhydrase (30000). soybean trypsin inhibitor (20 100) and cr-lactalbumin (14000): B = purified allergen.
NOTES
423
kg
OF ALLERGEN
Fig. 4. Titration of birch crude extract (m) and HPLC-purified main allergen (0). A pool of allergic sera (class 4 RAST) and 1251-labelled rabit anti-human IgE was used in SPRIA.
.-
pure allergen (lane B) showed a molecular weight value of 17 000 daltons, in agreement with the value detemined by HPLC. Immunological properties
To characterize further the purified allergen from an immunological view, we compared it with the crude extract in SPRIA.
point of
loo-
m l
a /
g
E . I af
* /
so-
. ./
l
-I
I
loo0
I 1W
I
10
I 1
RECIPROCAL OF ALLERGEN DILUTION
Fig. 5. RAST inhibition assay. The results are expressed as the percentage inhibition of binding of specific IgE achieved by addition of serial dilutions of allergen. The undiluted Pharmalgens extract corresponds to 100000 B.U./ml. n = HPLC-purified allergen: l = Pharmalgena extract.
424
NOTES
A dose-dependent binding of specific immunoglobin E (IgE), as revealed by the amount of r2+labelled rabbit antibody fixed to the wells, was obtained in both cases (Fig. 4). To reach 50% binding of radiactivity, 0.2 ,ng of the pure allergen and 1 pg of crude extract, are needed. Thus, the final specific activity of the purified protein is about five-fold greater than that of the starting material, in accord with the contribution of the allergen peak to the total amount of proteins (Fig. 1). The inhibition of RAST gave identical results to those obtained with the Pharmalgenm purified extract (Fig. 5). The same pool of sera from the patients used in Fig. 4 was used as a source of specific anti Betula IgE. 1000 Biological Units (B.U.) were required to achieve 50% inhibition in R&T, which corresponds to 1.6 pg of pure protein, as determined by the method of Lowry et a1.12. Hence, 1 B.U. is equivalent to 1.6 ng of pure B. verrucosa allergen. DISCUSSION
In this, the final communication of our recent work dealing with the purification of allergens from vegetal, fungal, mites or mammalian origin, we present the HPLC technology needed for a rapid and quantitative purification of the major allergen of birch pollen. As described previously J- lo, HPLC Protein Pack 125 columns and phosphate-buffered saline allow rapid separations of proteins in crude extracts as a function of their molecular weights. A specific immunoassay with sera from atopic patients provides us with a method for the localization of allergens in the fractions obtained after HPLC separation. The Be&la verrucosa main allergen is eiuted from the column in almost pure form in fraction 10 (Fig. 2). Its purity was checked by SDS-PAGE (Fig. 3) and its molecular weight, determined by HPLC and PAGE, corresponds with the values previously reported by other authors. The pure allergen, as is a rule in HPLC purification, retains its full immunological properties of IgE binding. This was demonstrated by titration against specific IgE-containing human sera (Fig. 4) and RAST inhibition (Fig. 5). The purified mollecule is enriched about. five-fold in comparison with a crude birch pollen extract, and we also determined that 1.6 ng are needed to produce 1 B.U. The industrial-scale purification of the allergen can provide allergic patients with a specific treatment instead of the semicrude-extract therapy currently used over the world. ACKNOWLEDGEMENT
The technical assistance of Miss Mercedes Alonso is gratefully acknowledged. REFERENCES 1 L. Belin, ht. Arch. Allergy Appl. Immunol., 42 (1972) 300. 2 M. Viander, J. Fraki, B. M. Djupsund and S. Laine, AI1erg.y.34 (1979) 289.
NOTES 3 4 5 6 7 8 9 10 11 12
J. Apold, E. Florvaag and S. Elsayed, Int. Arch. ANergy Appl. Immunol., 64 (1981) 439. H. Ipsen and H. Lowenstein, J. Allergy Clin. Zmmunol., 72 (1983) 150. A. Brieva and N. Rubio, J. Chromutogr., 370 (1986) 165. N. Rubio, A. Brieva and M. Rubio, J. Chromatogr., 392 (1987) 447. N. Rubio, A. Brieva and B. Alcazar, J. Chromatogr., 403 (1987) 312. N. Rubio and A. Brieva, J. Chromatogr., 404 (1987) 378. N. Rubio and A. Brieva, J. Chromatogr., 398 (1987) 366. N. Rubio and A. Brieva, J. Chromatogr., 407 (1987) 408. U. K. Laemmli, Nature (London), 227 (1970) 680. 0. H. Lowry, N. J. Rosebrough, A. L. Fan and R. L Randall, J. Biol. Chem., I93 (1951)
42.5
265.