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277 Mediator release in asthma after allergen inhalation, treadmill exercise, isocapnic hyperventilation and methacholine challenge
279
DETECTION OF ALTERNARIA (ALT) ALLERGENS BY WESTERN BLOTTING. L. Kroutil, B.S., R. Bush, M.D.. Madison. WI -Allergens present in ALT extracts have not been fully characterized. Polyacrylamide gel electrophroesis in the presence of the detergent sodium dodecyl sulfate (SDS-PAGE) allows high resolution of the proteins in ALT extracts and provides an estimation of their molecular weights, but does not permit the direct identification of IgE-binding proteins. used Western blotting to idenWe, therefore, tify allergens contained in ALT extracts which were first separated by SDS-PAGE. The ALT proteins were electrophoretically transferred out of SDS-PAGE gels into a nitrocellulose membrane, which was cut into strips for incubation with sera from ALT-sensitive and non-ALT-sensitive individuals. Washed strips were incubated with I125 labelled rabbit antihuman IgE and applied to x-ray film for autoradiography. Strips incubated with pooled sera from ALTsensitive patients and serum from one ALTsensitive individual showed radiostaining in the molecular weight region of 50-70,000 daltons (d). Another ALT-sensitive individserum showed a different pattern with ual's IgE binding to a 30,000 d. component. Strips incubated with sera from non-allergic or atopic, non-ALT sensitive individuals showed little or no radiostaining, indicating specific IgE binding. Thus, the technique of Western blotting following SDS-PAGE separation of ALT extracts permits both the characterization of relevant allergens and the specific IgE responses of ALT-sensitive patients.
280
ANALYSIS OF FUSARIUM SOLANI ANTIGENS/ALLERGENS. CE O'Neil, Ph.D., ML McCants, B.A., JE Salvaggio, M.D., and SB Lehrer, Ph.D., New Orleans, Louisiana Since antigen extracts of Fusarium $olani, a member of the Fungi Imuerfecti. are used routinely in clinical Allergy for diagnosis and treatment of mold-sensitive individuals; it is important to characterize them. Extracts of F. solani were prepared by sonicating powdered, defatted material in phosphate buffered saline extracting overnight at 4'C; (10 w/ml); centrifuging (48,000 g); concentrating (Amicon UM 2); and re-centrifuging (105,000 g). The final supernatant was fractionated by column chromatography (Bio-Gel A 0.5 m). Spectrophotometric analysis of column eluate (O.D. 230 & 280 nm) demonstrated the presence of 3 peaks of UV absorbing material corresponding to molecular weights of: >341,000 (Peak 1); 341,000 (Peak 2); and <42,000 (Peak 3). Fused rocket immunoelectrophoresis with rabbit antiF. solani demonstrated the presence of precipitating antigens in these peaks. Allergenic activity of column fractions was assessed by RAST inhibition and fused rocket radioimmunoelectrophoresis (FRRIE) using a pool of sera from individuals with F. solani specific IgE antibodies. Maximal RAST inhibition (43%) was associated with Peak 2, but was also present, to lesser degrees (23-27%) with Peaks 1 6 3. FRRIE showed maximal staining at Peak 1, with more diffuse staining at Peak 2 and essentially no staining at Peak 3. These studies demonstrated that F. solani contains several clinically relevant allergens which differ in molecular weight and charge.
Nine asthmatic subjects with hyperreactive airways (geometric mean PC20 methacholine 0.8 mg/ml) each underwent bronchial challenge with isocapnic hyperventilation allergen, exercise, (ISH) and methacholine, and had comparable falls in specific airways conductance (sGaw). None of the challenges were associated with any significant change in plasma histamine (including allergen inhalation in which only hyporeactive patients showed elevated levels). Serum NCA significantly increased after allergen (76.9 + 23.7%) and exercise (87.0 + 21.6%) but not methacholine (17.10 + 10.9%): With ISH, elevations in NCA were observed in 6 of the 9 subjects but as a group these increases were not statistically significant. The time course of NCA response differed between allergen and exercise, in that there was a sustained increase from 5 to 120 min post-allergen challenge, while the increase in NCA after exercise peaked at 10 min and then These results support the view that declined. both immunological and non-immunological mechanisms may trigger mediator release in asthma.
(BRIEF) investigate individual quantitate
procedure the
allergenic electrofocused
was
developed
to
allergenic activity IEF bands and to attempt the total allergenic activity Pollen extracts extracts. using
LKB-PAG
plates.
of to
the of
were
The IEF nitro human
were passively blotted onto paper, reacted with allergic IgE. radiolabelled anti-human Following exposure to radioautographic film. those bands that bind specific IgE were The BRIEF templates were then cut revealed. The BRIEF and counted in a gamma counter. technique was employed to visualize and quantitate the relative importance of Antigen E activity in a Short Ragweed extract, using In two sera from 6 allergic patients. patients, Antigen E accounted for more than 757. of the total counts bound by Short in the other 4 serum Ragweed extract; samples, Antigen E was responsible for BRIEF between 38 and 54% of the activity. autoradiograms of June, Rye, Timothy, Bermuda, Short and Giant Ragweed extracts using RAST positive serum pools for Ragweeds, Bermuda and Grasses demonstrated allergens in and the patterns were unique all pH areas, The Grass and Bermuda far each extract. serum pools showed little activity with the Ragweed extracts, yet different patter;;eof specificity with individual grasses. BRIEF technique is useful in examining the Standardized Rye comparabi li ty of extracts. extracts bound approximately 80% of the activity of the U.S. Reference. Nonstandardized rye extracts were found to bind more variable amounts of activity, and generally less relative activity than the BRIEF provides a standardized preparations. method of comparing reference extracts and serum pools for the presence of individual allergens without animal set-a or activated and has the potential for supports. quantitatively determining the relative potency of allergenic extracts.
patterns
cellulose 5era,
and
174
DETECTION OF ALTERNARIA (ALT) ALLERGENS BY WESTERN BLOTTING. L. Kroutil, B.S., R. Bush, M.D.. Madison. WI -Allergens present in ALT extracts have not been fully characterized. Polyacrylamide gel electrophroesis in the presence of the detergent sodium dodecyl sulfate (SDS-PAGE) allows high resolution of the proteins in ALT extracts and provides an estimation of their molecular weights, but does not permit the direct identification of IgE-binding proteins. used Western blotting to idenWe, therefore, tify allergens contained in ALT extracts which were first separated by SDS-PAGE. The ALT proteins were electrophoretically transferred out of SDS-PAGE gels into a nitrocellulose membrane, which was cut into strips for incubation with sera from ALT-sensitive and non-ALT-sensitive individuals. Washed strips were incubated with I125 labelled rabbit antihuman IgE and applied to x-ray film for autoradiography. Strips incubated with pooled sera from ALTsensitive patients and serum from one ALTsensitive individual showed radiostaining in the molecular weight region of 50-70,000 daltons (d). Another ALT-sensitive individserum showed a different pattern with ual's IgE binding to a 30,000 d. component. Strips incubated with sera from non-allergic or atopic, non-ALT sensitive individuals showed little or no radiostaining, indicating specific IgE binding. Thus, the technique of Western blotting following SDS-PAGE separation of ALT extracts permits both the characterization of relevant allergens and the specific IgE responses of ALT-sensitive patients.
280
ANALYSIS OF FUSARIUM SOLANI ANTIGENS/ALLERGENS. CE O'Neil, Ph.D., ML McCants, B.A., JE Salvaggio, M.D., and SB Lehrer, Ph.D., New Orleans, Louisiana Since antigen extracts of Fusarium $olani, a member of the Fungi Imuerfecti. are used routinely in clinical Allergy for diagnosis and treatment of mold-sensitive individuals; it is important to characterize them. Extracts of F. solani were prepared by sonicating powdered, defatted material in phosphate buffered saline extracting overnight at 4'C; (10 w/ml); centrifuging (48,000 g); concentrating (Amicon UM 2); and re-centrifuging (105,000 g). The final supernatant was fractionated by column chromatography (Bio-Gel A 0.5 m). Spectrophotometric analysis of column eluate (O.D. 230 & 280 nm) demonstrated the presence of 3 peaks of UV absorbing material corresponding to molecular weights of: >341,000 (Peak 1); 341,000 (Peak 2); and <42,000 (Peak 3). Fused rocket immunoelectrophoresis with rabbit antiF. solani demonstrated the presence of precipitating antigens in these peaks. Allergenic activity of column fractions was assessed by RAST inhibition and fused rocket radioimmunoelectrophoresis (FRRIE) using a pool of sera from individuals with F. solani specific IgE antibodies. Maximal RAST inhibition (43%) was associated with Peak 2, but was also present, to lesser degrees (23-27%) with Peaks 1 6 3. FRRIE showed maximal staining at Peak 1, with more diffuse staining at Peak 2 and essentially no staining at Peak 3. These studies demonstrated that F. solani contains several clinically relevant allergens which differ in molecular weight and charge.
Nine asthmatic subjects with hyperreactive airways (geometric mean PC20 methacholine 0.8 mg/ml) each underwent bronchial challenge with isocapnic hyperventilation allergen, exercise, (ISH) and methacholine, and had comparable falls in specific airways conductance (sGaw). None of the challenges were associated with any significant change in plasma histamine (including allergen inhalation in which only hyporeactive patients showed elevated levels). Serum NCA significantly increased after allergen (76.9 + 23.7%) and exercise (87.0 + 21.6%) but not methacholine (17.10 + 10.9%): With ISH, elevations in NCA were observed in 6 of the 9 subjects but as a group these increases were not statistically significant. The time course of NCA response differed between allergen and exercise, in that there was a sustained increase from 5 to 120 min post-allergen challenge, while the increase in NCA after exercise peaked at 10 min and then These results support the view that declined. both immunological and non-immunological mechanisms may trigger mediator release in asthma.
(BRIEF) investigate individual quantitate
procedure the
allergenic electrofocused
was
developed
to
allergenic activity IEF bands and to attempt the total allergenic activity Pollen extracts extracts. using
LKB-PAG
plates.
of to
the of
were
The IEF nitro human
were passively blotted onto paper, reacted with allergic IgE. radiolabelled anti-human Following exposure to radioautographic film. those bands that bind specific IgE were The BRIEF templates were then cut revealed. The BRIEF and counted in a gamma counter. technique was employed to visualize and quantitate the relative importance of Antigen E activity in a Short Ragweed extract, using In two sera from 6 allergic patients. patients, Antigen E accounted for more than 757. of the total counts bound by Short in the other 4 serum Ragweed extract; samples, Antigen E was responsible for BRIEF between 38 and 54% of the activity. autoradiograms of June, Rye, Timothy, Bermuda, Short and Giant Ragweed extracts using RAST positive serum pools for Ragweeds, Bermuda and Grasses demonstrated allergens in and the patterns were unique all pH areas, The Grass and Bermuda far each extract. serum pools showed little activity with the Ragweed extracts, yet different patter;;eof specificity with individual grasses. BRIEF technique is useful in examining the Standardized Rye comparabi li ty of extracts. extracts bound approximately 80% of the activity of the U.S. Reference. Nonstandardized rye extracts were found to bind more variable amounts of activity, and generally less relative activity than the BRIEF provides a standardized preparations. method of comparing reference extracts and serum pools for the presence of individual allergens without animal set-a or activated and has the potential for supports. quantitatively determining the relative potency of allergenic extracts.
patterns
cellulose 5era,
and
174