- Email: [email protected]
Cross allergenicity among oleaceae pollens
209 CYTOCHROME C AS A POSSIBLE ALLERGEN IN BERMUDA
211 CROSS ALLERGENICITY AMONGOLEACEAE POLLENS. d. Bousquet,M.D., B. Gu6rin,Ph.D., H. Dhivert~ M.D., B. Hewitt, F.B. Michel,M.D., Montpellier, and Joinville-Le-Pont, France.
GRASS POLLEN EXTRACT. Michael J Schumacher, MB~ FRACP~ Carmen M Wagner~ PhD~ and Michael A Cusanovich, PhD, Tucson, Arizona Bermuda grass pollen, one of the most important allergens in warm climate areas, was extracted and fractionated to identify major allergenic components. A combination of carboxymethylcellulose (CM-C) and diethylaminoethyl cellulose (DEAE-C) chromatography was used, followed by Sephadex G-75. Specific activity of material in each peak was estimated by endpoint skin testing of allergic volunteers. After extraction of 50 gm of defatted Bermuda grass pollen in 0.i M bicarbonate buffer, pH 8.0, inactive low molecular weight material was removed by Sephadex G-25 chromatography. The material was then equilibrated with a 5 mM phosphate buffer, pH 6.0 and applied to a CM-C column. The CM-C bypass material was used for subsequent fractionation on DEAE-C. Components bound to the CM-C were eluted with a stepwise NaCI gradient (20-80 mM NaCI) in 5 mM phosphate buffer pH 6.0. The material eluted at 40 and 60 mM NaCI was recycled through CM-C and found to be allergenic and rich in cytochrome C, as suggested by analytical studies and absorption spectra. CM-C bypass material was equilibrated at pH 7.5, applied to, and eluted from a DEAE-C column with a NaCI gradient (5 mM - 0.5 M NaCI). Potent fractions were eluted at < 0.25 M and were found to lie in the fractionation range of Sephadex G-75. It was concluded that Bermuda grass pollen contains several major allergens, one of which is associated with cytochrome C.
Pollens of related species usually have several cross-reacting antigens although this is not always the case. In a preliminary report we have shown that pollens of the Oleaceae family ( o l i v e , ash, p r i v e t , Phillyrea) have several cross-reacting antigens in rats. The crossa l l e r g e n i c i t y among these four Oleaceae species was further tested by means of RAST-inhibition and i s o - e l e c t r i c focusing (IEF). RAST-inhibition experiments were performed using 4 d i f f e r e n t antigens coupled to paper discs (4 pollen species) and for each experiment a pool of 6 sera of patients highly a l l e r g i c to Oleaceae pollens was used. The results show a high degree of i n h i b i t i o n of the RAST whatever allergen coupled to the disc or allergen used for the i n h i b i t i o n of the test. Since RAST estimates total allergenic potency only, protein p r o f i l e s of each pollen species were examined by IEF and compared with that of a timothy pollen extract. Most proteins of the 40leaceae species were in the acidic range of pH (pH3 to 4.2) whereas for grass pollen the f i r s t protein to appear was at pH 4.1 and most proteins are found in the range between pH 4.3 to 5.1. For each species, however, there appear a few proteins in the neutral and basic range of pH. Furthermore, there are fewer differences between Oleaceae pollens than between grass pollens. The IEF experiments confirmed the high degree of s i m i l a r i t y between Oleaceae pollens.
210 I~UNOCHEMICAL
212 THE ISOLATION OF A UNIQUE BASIC PEPTIDE ALLER-
CHARACTERIZATION OF A PURIFIED ALLERGEN FROM RYE GRASS (LOLIUM PERENNE) POLLEN. A.K.M. Ekramoddoulah, F~ Kisil, and A.H. Sehon. MRC Group for Allergy Research. Immunology Dept., Univ. of Manitoba, Winnipeg, Canada. A high molecular weight basic allergen (HMBA) was isolated from the mixture of nondialysable components of the aqueous extract of defatted rye grass pollen by a combination of gel filtration and isoelectrofoeussing. HMBA, a glycoprotein of 57,000 daltons (~ 17% carbohydrate) contained all naturally occurring amino acids. It migrated as a single component in PAGE and in SDS-PAGE. A hyperimmune rabbit anti-HMBA serum gave only a single precipitin band with the crude extract of the rye grass pollen in crossed immunoelectrophoresis. Thus, it was concluded that HMBA was a unique and highly purified antigen. HMBA elicited a strong IgE antibody response in C3H/HeJ mice. Denaturation of HMBA by 7M guanidine HCI led to the loss of allergenicity (i.e. inability to elicit PCA reactions with the murine reaginie antiserum to HMBA) and antigenicity (inability to form precipitins with rabbit anti-HMBA serum); hence, it would appear that the allergenic and antigenic determinants of HMBA are "conformational". The allergenieity of HMBA was also established by direct skin tests (kindly performed by Dr. R. Warrington) in grass sensitive patients and by RAST utilizing HMBA discs. The allergenic cross reactivity bet w e e n H M B A and the other constituents of crude rye grass pollen extract is being established by RAST inhibition.
GEN FROM ALTERNARIA EXTRACTS. T.W. Budd, Ph.D., C.Y. Kuo, Ph.D.~ T.J. Yoo, M.D., Ph.D,~ Memphis~ TN~ J. Cazin, Ph.D., Iowa City, Iowa. A basic peptide allergen has been isolated from Alternaria extracts. Flat bed gel preparitive isoelectric focusing (IEF) followed by dialysis and lyophilization allowed the concentration of substantial quantities of this heretofore unrecognized allergen. Rocket immunoelectrophoresis autoradiography (usin~ RAST Class 3 Alternaria patient sera and 12~lanti HuIgE) and a RAST inhibition assay were used to demonstrate the allergenicity of this fraction. SDS-PAGE was used to determine homogeneity and approximate molecular weight. The IEF fraction banded at a pH of 9.5 - 9.8 . SDS-PAGE showed the fraction contained two bands (one major and one ~inor) which migrated between the ion front and the broraophenol blue tracking dye. Calibration proteins were used with the smallest (Lysozyme-14000 MW) banding at the position of the tracking dye, thus indicating a molecular weight of under 14,000 daltons for the allergen fraction~ The allergenicity of this protein was sufficient to give 50% RAST inhibition at a concentration of 400 ~g/ml. The im~unoelectrophoresis autoradiography also indicated allergenicity. To the best of our knowledge, this is the first time that a basic peptide has been sho~n to be allergenic. This is but one of several major allergens of Alternaria to be isolated and characterized for the purpose of future standardization of extracts used for diagnosis and treatment of mold allergy.
145
211 CROSS ALLERGENICITY AMONGOLEACEAE POLLENS. d. Bousquet,M.D., B. Gu6rin,Ph.D., H. Dhivert~ M.D., B. Hewitt, F.B. Michel,M.D., Montpellier, and Joinville-Le-Pont, France.
GRASS POLLEN EXTRACT. Michael J Schumacher, MB~ FRACP~ Carmen M Wagner~ PhD~ and Michael A Cusanovich, PhD, Tucson, Arizona Bermuda grass pollen, one of the most important allergens in warm climate areas, was extracted and fractionated to identify major allergenic components. A combination of carboxymethylcellulose (CM-C) and diethylaminoethyl cellulose (DEAE-C) chromatography was used, followed by Sephadex G-75. Specific activity of material in each peak was estimated by endpoint skin testing of allergic volunteers. After extraction of 50 gm of defatted Bermuda grass pollen in 0.i M bicarbonate buffer, pH 8.0, inactive low molecular weight material was removed by Sephadex G-25 chromatography. The material was then equilibrated with a 5 mM phosphate buffer, pH 6.0 and applied to a CM-C column. The CM-C bypass material was used for subsequent fractionation on DEAE-C. Components bound to the CM-C were eluted with a stepwise NaCI gradient (20-80 mM NaCI) in 5 mM phosphate buffer pH 6.0. The material eluted at 40 and 60 mM NaCI was recycled through CM-C and found to be allergenic and rich in cytochrome C, as suggested by analytical studies and absorption spectra. CM-C bypass material was equilibrated at pH 7.5, applied to, and eluted from a DEAE-C column with a NaCI gradient (5 mM - 0.5 M NaCI). Potent fractions were eluted at < 0.25 M and were found to lie in the fractionation range of Sephadex G-75. It was concluded that Bermuda grass pollen contains several major allergens, one of which is associated with cytochrome C.
Pollens of related species usually have several cross-reacting antigens although this is not always the case. In a preliminary report we have shown that pollens of the Oleaceae family ( o l i v e , ash, p r i v e t , Phillyrea) have several cross-reacting antigens in rats. The crossa l l e r g e n i c i t y among these four Oleaceae species was further tested by means of RAST-inhibition and i s o - e l e c t r i c focusing (IEF). RAST-inhibition experiments were performed using 4 d i f f e r e n t antigens coupled to paper discs (4 pollen species) and for each experiment a pool of 6 sera of patients highly a l l e r g i c to Oleaceae pollens was used. The results show a high degree of i n h i b i t i o n of the RAST whatever allergen coupled to the disc or allergen used for the i n h i b i t i o n of the test. Since RAST estimates total allergenic potency only, protein p r o f i l e s of each pollen species were examined by IEF and compared with that of a timothy pollen extract. Most proteins of the 40leaceae species were in the acidic range of pH (pH3 to 4.2) whereas for grass pollen the f i r s t protein to appear was at pH 4.1 and most proteins are found in the range between pH 4.3 to 5.1. For each species, however, there appear a few proteins in the neutral and basic range of pH. Furthermore, there are fewer differences between Oleaceae pollens than between grass pollens. The IEF experiments confirmed the high degree of s i m i l a r i t y between Oleaceae pollens.
210 I~UNOCHEMICAL
212 THE ISOLATION OF A UNIQUE BASIC PEPTIDE ALLER-
CHARACTERIZATION OF A PURIFIED ALLERGEN FROM RYE GRASS (LOLIUM PERENNE) POLLEN. A.K.M. Ekramoddoulah, F~ Kisil, and A.H. Sehon. MRC Group for Allergy Research. Immunology Dept., Univ. of Manitoba, Winnipeg, Canada. A high molecular weight basic allergen (HMBA) was isolated from the mixture of nondialysable components of the aqueous extract of defatted rye grass pollen by a combination of gel filtration and isoelectrofoeussing. HMBA, a glycoprotein of 57,000 daltons (~ 17% carbohydrate) contained all naturally occurring amino acids. It migrated as a single component in PAGE and in SDS-PAGE. A hyperimmune rabbit anti-HMBA serum gave only a single precipitin band with the crude extract of the rye grass pollen in crossed immunoelectrophoresis. Thus, it was concluded that HMBA was a unique and highly purified antigen. HMBA elicited a strong IgE antibody response in C3H/HeJ mice. Denaturation of HMBA by 7M guanidine HCI led to the loss of allergenicity (i.e. inability to elicit PCA reactions with the murine reaginie antiserum to HMBA) and antigenicity (inability to form precipitins with rabbit anti-HMBA serum); hence, it would appear that the allergenic and antigenic determinants of HMBA are "conformational". The allergenieity of HMBA was also established by direct skin tests (kindly performed by Dr. R. Warrington) in grass sensitive patients and by RAST utilizing HMBA discs. The allergenic cross reactivity bet w e e n H M B A and the other constituents of crude rye grass pollen extract is being established by RAST inhibition.
GEN FROM ALTERNARIA EXTRACTS. T.W. Budd, Ph.D., C.Y. Kuo, Ph.D.~ T.J. Yoo, M.D., Ph.D,~ Memphis~ TN~ J. Cazin, Ph.D., Iowa City, Iowa. A basic peptide allergen has been isolated from Alternaria extracts. Flat bed gel preparitive isoelectric focusing (IEF) followed by dialysis and lyophilization allowed the concentration of substantial quantities of this heretofore unrecognized allergen. Rocket immunoelectrophoresis autoradiography (usin~ RAST Class 3 Alternaria patient sera and 12~lanti HuIgE) and a RAST inhibition assay were used to demonstrate the allergenicity of this fraction. SDS-PAGE was used to determine homogeneity and approximate molecular weight. The IEF fraction banded at a pH of 9.5 - 9.8 . SDS-PAGE showed the fraction contained two bands (one major and one ~inor) which migrated between the ion front and the broraophenol blue tracking dye. Calibration proteins were used with the smallest (Lysozyme-14000 MW) banding at the position of the tracking dye, thus indicating a molecular weight of under 14,000 daltons for the allergen fraction~ The allergenicity of this protein was sufficient to give 50% RAST inhibition at a concentration of 400 ~g/ml. The im~unoelectrophoresis autoradiography also indicated allergenicity. To the best of our knowledge, this is the first time that a basic peptide has been sho~n to be allergenic. This is but one of several major allergens of Alternaria to be isolated and characterized for the purpose of future standardization of extracts used for diagnosis and treatment of mold allergy.
145