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397 Partial purification of an alternaria (ALT) allergen by electroelution
395
396
IMMDNOPRINTANALYSIS OF CALVATIA CYATHIFOBMIS (C.C.) BASIDIOSPORE ALLERGENS: II. TIME AND TEMPERATURESTABILITY. MD Ibanez, MD, WE Horner, PhD, and SB Lehrer, PhD, New Orleans, LA. C.C. allergens in unfractionated extract (cr;dg), and sequentially fractionated by gel filtration (GF) and hydrophobic interaction chromatography (HIC), were tested for stability. sources (crude, GF, HIC) were --C.C. allergen resuspended in 1% glycine (pH 6.5, 20 mg/ml), sampled immediately (0 hour), aliquotted and incubated at 4OC, 24'C, and 37'C. Incubations were sampled at 8, 24 and 96 hr. Samples, snap frozen and stored (less than 2 wk) at -80°C, were PAGE-electrofocused in duplicate for immunoprinting and staining. Immunoprints incubated in a sera pool 5 C.C. BAST positive patients), reacted with 1I 51?;beled anti-IgE and autoradiographed, revealed 3 allergen(s) groups, or bands (Bd) with respective pI of 3,6-4.6, 6.7 and 9.5. Only Bds3.6-4.6 were stable at 37OC. At 24'C, Bds3.6-4.6 persisted to 96 hr, Bd6.7 persisted at 24 hr, and Bd9.5 waned in 8 hr. At 4'C all 3 allergens in HIC were stable for 8 and 24 hr; Bd9.5 was reduced at 96 hr. These stability patterns corresponded to the stained gels and were consistent among different sources. Bd9.5 is very labile, but reactive with 63% of sera tested. Since lo-15% of C.C. reactors bind IgE only to Bd9.5, this varTaFility is notable and significant for diagnosis and treatment.
397
We have previously identified glyoprotein with a molecular weight of 66 K: as an important allergen in extracts prepared 46582 (JACI 80:170, from ALT, ATCC strain We sought to purify thisxergen by 1987). ALT extract was electroelution. Crude resolved on 12.5% SDS-polyacrylamide gels at 6OmA for 3 hours. Portibns of the gel corresponding to the 66 Kd region were electroeluted at 3 Watts for 4 hours. Electroeluted samples concentrated, were Thirty ug of the lyophylized, and pooled. pooled sample were resolved by SDS-PAGE. Coomassie blue staining of the gel revealed a single
band with
an apparent
66 Kd while silver
staining
molecular
wt. of
detected 2 bands
in this region. Imnunoblotting demonstrated IgE binding to this allergen using sera frcan
ALT-sensitive subjects and prick skin tests gave positive reactions in ALT-sensitive patients. We conclude that electroelution is a useful, single step technique for the purification
of
ALT and
potentially
other
allergens.
393
Characterization of Major and Eypo-Allergens of Eari U. Vijay, Ph.D., Alternaria Tenuis. Maureen Burton, B.Sc., and 19. Martin Young, Ph.D., Ottawa, Canada. Extracts of A. tenuis contain in addition to the major allergens, hypoallergenic antigens value. of potential immunotherapeutic w immunoblots of SDS-PAGE gels showed strong bands from the major allergen at 59,000, 77,000 and 94,000 y and the hypoallergen at 16,000 and 18,000 MW together with minor components at To characterize these 30,000 and 42,000 MU. components, an A. tenuis extract was separated by gel-filtratgn on Sephadex G-100. The fractions containing the major allergens were identified by their high activity in RAST The bands seen in inhibition and PCA tests. SDS-PAGE immunoblots between 50,000-100,000 were present in these fractions. In isoelectric focusing four bands with pIs between 3.75 and 4.15 were found. A second group of fractions, active in RAST inhibition but not active in PCA, was identified as the hypoallergen component. These fractions showed the 16,000 and 18,000 MW bands and the weslcer 30,000 MW band. They eluted from the Sephadex column with an apparent MU of 30,000, however. In IEF, there were 4 bands in the 4.15 to 4.55 p1 region. The hypoallergen was further purified by ionexchange chromatography, and characterized by amino acid and sugar analyses. The antigenic behavior of the allergen and the hypoallergen show that they are extensively cross-reactive. Hence, it is probable that the hypoallergenic components are fragments of the allergens.
267
ALTERNARIA
ALTERWATA
PURE SPORE EXTRACTS
(PROTEIN CONTENT, BIOLOGIC ACTIVITY AND STABILITY) John Santilli, Jr., M.D., William J. Rockwell, M.D., Ralph P. Collins, Ph.D., and Jan Narciso, Ph.D. Bridgeport, CT and Storrs, CT Three lots of spore extracts of Alternaria alternata prepared 2 years apart were studied for protein content, biologic activity and stability. Ninhydrin analysis on a 1:40 wt/vol extract in 50% glycerine of each lot was as follows: lot 1: 3754ug/ml, lot 2: 3640 ug/ml, lot 3: 1496ug/ml. All three lots were diluted so that each contained 800ug/ml ninhydrin protein. A RAST inhibition assay to determine the relative potency of each lot was performed with the following results. Using lot 1 as the standard, the relative potency of lot 2 was llO%, and the relative potency of lot 3 was 95%. This study shows that there is lot to lot variation in 1:40 wt/vol extracts. Good correlation was shown between extracts containing 800ug/ml of ninhydrin protein and RAST testing. These studies have also shown that Alternaria extracts stored in 50% glycerine are stable for at least four years.
396
IMMDNOPRINTANALYSIS OF CALVATIA CYATHIFOBMIS (C.C.) BASIDIOSPORE ALLERGENS: II. TIME AND TEMPERATURESTABILITY. MD Ibanez, MD, WE Horner, PhD, and SB Lehrer, PhD, New Orleans, LA. C.C. allergens in unfractionated extract (cr;dg), and sequentially fractionated by gel filtration (GF) and hydrophobic interaction chromatography (HIC), were tested for stability. sources (crude, GF, HIC) were --C.C. allergen resuspended in 1% glycine (pH 6.5, 20 mg/ml), sampled immediately (0 hour), aliquotted and incubated at 4OC, 24'C, and 37'C. Incubations were sampled at 8, 24 and 96 hr. Samples, snap frozen and stored (less than 2 wk) at -80°C, were PAGE-electrofocused in duplicate for immunoprinting and staining. Immunoprints incubated in a sera pool 5 C.C. BAST positive patients), reacted with 1I 51?;beled anti-IgE and autoradiographed, revealed 3 allergen(s) groups, or bands (Bd) with respective pI of 3,6-4.6, 6.7 and 9.5. Only Bds3.6-4.6 were stable at 37OC. At 24'C, Bds3.6-4.6 persisted to 96 hr, Bd6.7 persisted at 24 hr, and Bd9.5 waned in 8 hr. At 4'C all 3 allergens in HIC were stable for 8 and 24 hr; Bd9.5 was reduced at 96 hr. These stability patterns corresponded to the stained gels and were consistent among different sources. Bd9.5 is very labile, but reactive with 63% of sera tested. Since lo-15% of C.C. reactors bind IgE only to Bd9.5, this varTaFility is notable and significant for diagnosis and treatment.
397
We have previously identified glyoprotein with a molecular weight of 66 K: as an important allergen in extracts prepared 46582 (JACI 80:170, from ALT, ATCC strain We sought to purify thisxergen by 1987). ALT extract was electroelution. Crude resolved on 12.5% SDS-polyacrylamide gels at 6OmA for 3 hours. Portibns of the gel corresponding to the 66 Kd region were electroeluted at 3 Watts for 4 hours. Electroeluted samples concentrated, were Thirty ug of the lyophylized, and pooled. pooled sample were resolved by SDS-PAGE. Coomassie blue staining of the gel revealed a single
band with
an apparent
66 Kd while silver
staining
molecular
wt. of
detected 2 bands
in this region. Imnunoblotting demonstrated IgE binding to this allergen using sera frcan
ALT-sensitive subjects and prick skin tests gave positive reactions in ALT-sensitive patients. We conclude that electroelution is a useful, single step technique for the purification
of
ALT and
potentially
other
allergens.
393
Characterization of Major and Eypo-Allergens of Eari U. Vijay, Ph.D., Alternaria Tenuis. Maureen Burton, B.Sc., and 19. Martin Young, Ph.D., Ottawa, Canada. Extracts of A. tenuis contain in addition to the major allergens, hypoallergenic antigens value. of potential immunotherapeutic w immunoblots of SDS-PAGE gels showed strong bands from the major allergen at 59,000, 77,000 and 94,000 y and the hypoallergen at 16,000 and 18,000 MW together with minor components at To characterize these 30,000 and 42,000 MU. components, an A. tenuis extract was separated by gel-filtratgn on Sephadex G-100. The fractions containing the major allergens were identified by their high activity in RAST The bands seen in inhibition and PCA tests. SDS-PAGE immunoblots between 50,000-100,000 were present in these fractions. In isoelectric focusing four bands with pIs between 3.75 and 4.15 were found. A second group of fractions, active in RAST inhibition but not active in PCA, was identified as the hypoallergen component. These fractions showed the 16,000 and 18,000 MW bands and the weslcer 30,000 MW band. They eluted from the Sephadex column with an apparent MU of 30,000, however. In IEF, there were 4 bands in the 4.15 to 4.55 p1 region. The hypoallergen was further purified by ionexchange chromatography, and characterized by amino acid and sugar analyses. The antigenic behavior of the allergen and the hypoallergen show that they are extensively cross-reactive. Hence, it is probable that the hypoallergenic components are fragments of the allergens.
267
ALTERNARIA
ALTERWATA
PURE SPORE EXTRACTS
(PROTEIN CONTENT, BIOLOGIC ACTIVITY AND STABILITY) John Santilli, Jr., M.D., William J. Rockwell, M.D., Ralph P. Collins, Ph.D., and Jan Narciso, Ph.D. Bridgeport, CT and Storrs, CT Three lots of spore extracts of Alternaria alternata prepared 2 years apart were studied for protein content, biologic activity and stability. Ninhydrin analysis on a 1:40 wt/vol extract in 50% glycerine of each lot was as follows: lot 1: 3754ug/ml, lot 2: 3640 ug/ml, lot 3: 1496ug/ml. All three lots were diluted so that each contained 800ug/ml ninhydrin protein. A RAST inhibition assay to determine the relative potency of each lot was performed with the following results. Using lot 1 as the standard, the relative potency of lot 2 was llO%, and the relative potency of lot 3 was 95%. This study shows that there is lot to lot variation in 1:40 wt/vol extracts. Good correlation was shown between extracts containing 800ug/ml of ninhydrin protein and RAST testing. These studies have also shown that Alternaria extracts stored in 50% glycerine are stable for at least four years.