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187 Cross reactivity between Olea europea L. (OE), Syringa vulgaris L. (SV) and Fraxinus excelsior L. (FE)
VOLUME NUMBER
185
Abstracts
87 1. PART 2
TIME OF ELUTION-DEPENDENT ELECTROPHORETIC PATTERNS OF ALTERNARIA (ALT) GLYCOPROTEINS. c Barnes PhD. F Pachcco MS. J Portnov MD. Kansas City, Missouri. Many factors contribute to the content of ALT extracts including the kinetics of glycoprotein release during extraction. To qualitatively and quantitatively demonstrate the differences between quickly and slowly &ted materials, the following study was pcrformcd. Four strains of ALT were grown for 4 weeks in defined medium under identical conditions. The fungal mats were air-dried for 24 hours, powdered with mortar and pestle, and extracted at 4°C with stirring in distilled water (DW). During extraction, all of the supcrnate was removed at 1, 3. 6, and 24 hours and was replaced with an equal volume of DW. Extraction of undissolved materials then continued during the next time interval. All preparations were dialysed YS50 mM NH4HC03 and lyophilized. Reconstitution was in DW at lOmg/ml (w/v). Protein concentrations were measuredby Lowry, and carbohydrate by the phenol-sulfuric acid method. Preparations were subjected to SDS-PAGE and analylical agarosc IEF. Western blots of the SDS-PAGE were probed for IgE binding with pooled scra from ALT-sensitive patients. Extrachblc dry weights decreaxd with each iltnc ii:icrval while carbohydrate to protein ratio increased. SDS-PAGE rcvcalcd similar bands bciwccn strains at all times, most pron~incntly at 1’7.40. and 70kD. The 17kD band appears in 3 strains at 1, 3. and G hours. but not at 24 hours. The 40 and 70kD bands wcrc notable only al 6 and 24 hours. On IEF. a band at pI 3.5 which corrclatcs with a 70kD plycoprotcin previously isolntcd from ALT by a monoclonal antibody. was mom prominent at 24 hours. Significant IgE-rccognizcd bands were seen at 35. 50.60, and 135kD. The 35kD band was primxily dctcctcd at 1 hour. The 50 and 60 kD ones were seen at 24hrs in 2 strains and didn’t vary with time in the other 2. The 135kD band was only seen at 24hrs in 2 strains. Minor bands were obscrvcd at 17 and 70kD and wcrc also time dcpcndcnt. Release of individual glycoproteins from ALT occurs at dilfcrcnt rates during clution. Several of these are uniquely present in prcparations eluted for specific time intervals. Therefore, the elution time must be optimized if production of preparations containing any particular combination of glycoprotcins is desired.
186 EFFECTS OF AIR POLLUTANTS ON HAYFEVER SYMPTOM SEVERlTY G. Gunner. M.Sc. Greta k Rilev. BSc., Penny Fitzharris. M.D. and M.R. &hmore.Ph.D., London, U.K. We investigated the influence of ambient air pollutants on the severity of hayfever symptoms in a group of 76 patients who lived and worked in central London during the 1990U.K. grasspollen smscm. Symptom scores and medication use were recorded daily for the entire seasonand data for NO2, NO, 03 and SO2 concentrations obtained from London monitoring sites. Daily central London pollen counts were also obtained. After fitting a model basedon pollen counts the extent to which the residual variation in daily symptomscould be explained hy air pollutants was examined. Although the daily pollen counts show4 a significant correlation with symptomsscore, a modified pollen count, Pmod, which allowed a lag effect from previous days’ exposure significantly improved the tit to the data. With the addition of the cumulative pollen count, Pcum,60% of the variation in mean daily symptom score (S) could be explained by a model of the form S = aI + a2lnPmod + a3lnPcum. The addition of NO2 concentrations to the model further reduced the residual variation and significantly improved the fit to the data. Other pollutants and climatic variables showed no significant effects. These results suggestthat a relatively simple model provides a good fit to the observed symptom data and, furthermore, that ambient NO2 may contribute significantly to hayfever symptom severity in London.
185
187
CROSS RFACTISYRINGA VULGARIS L. (SV),AND FRAXINUS EXCELSI;IR L. (FE): ' 4. Wahl PhD, L J. fEoig BA, L M. Wimnel BA, " A. Caneaero MD ' D-2057 Reinbek (FRG). L Barcelone. ti Sevilia (Soain) Sensibilisation-to OE-pollen~;s v&-y common in the mediterranean area. SV and FE belong to the same family as OE. We investigated the cross reactivity between the three species. The sera of 12 OE-pollen allergic patients were investigated with different techniques. Skin tests were positive with the three extracts. The strongest reaction could be seen with OE. Using allergen disks, loaded with the three pollen extracts, positive RAST-classes (R-cl) could be found. The highest could be measured with OE-allergen disks (mean R-cl 3.6). With FE and SV disks RAST-class 2.7 (mean) was measured. Using the poolserum (N=12), the three kinds of allergen disks and the three extracts (vice versa), cross reactivity of different degrees could be found by RAST-inhibition. IEF and SDS-PAGE of all extracts showed complex protein patterns. IEF determined proteins were in an IP-range between 3.7 and 5.4. By SDS-PAGE most of them could be detected in a molecular weight range between 14.4 and 94.0 kD. The immunoprint showed most of the comnon allergens in an Ip-range between 4.5 and 5.4 and westernblots showed most of the common allergens with molecular weights between 14.4 and 20.0 kD. By these blotting techniques also species-specific allergens could be detected in the three extracts. Our results show a high degree of cross reactivity between OE, FE and SV. This should be taken into account if all three species are present in the local flora.
188
mMUNOCHEMICALANALYSIS
OF BAHIA GBBss cr. w. L. TNdeau. B.A. (MOD). E. F-s Ph.D.. D. K. Ledford. M.D.. G. A. Bucholtz. M.D. and R. F. Lockev M.D.& Tampa, FL. Sensitization to Bahia grass pollen (BGP). a common United States, has been grass in the Southern documented by skin testing, RAST and nasal challange. The allergenic composition of BGP was studied by preparative scale IEF. SDS-PAGE, IEF electrophoresis. RAST and immunoblot. Twenty grams of BGP were in ammonium defatted, extracted (1 : 20 w/v) bicarbonate, dialyzed and centrifuged. Sixty ml of the supemantant (12’3 mg protein) were loaded into a with 2% ampholyte, pH 3.5-10, and Rotofor@ electrophoresed for 6 hours at 12 W. Twenty fractions were harvested. Coomassie blue staining of the IEF gel showed bands in the pH range of 4.0-9.1. RAST to the individual fractions using 6 sera demonstrated consistent IgE binding in the pH range of 4.65 to 10. Higher IgE binding was detected to 7 fractions in the pH range of 4.5-6.5 and IO 6 fractions in the pH range of 7.3-10. Immunoblots revealed 1 major IgE binding band in the acidic fractions (AF) at 31 kD and 2 bands in the basic fractions (BF) at 33 kD and 14 kD. Pooled AF (78.8 mg) and BF $6.8 mg) were electrophoresed and examined by RAST. IgE sparately in the Rotofor binding to AF remained high in the pH range 4.5-6.9 and to BF in the pH range 7.7-9.5. Fractions in these pH ranges were pooled, dialyzed, lyophylized and chromatographed on a Sephadex G-50 column. Both AF and BF eluted in two peaks. BGP has three major allergens with molecular weights of 31 kD, 33 kD and 14 kD which occur in the pH ranges of 4.5-6.5 and 7.3-10.
185
Abstracts
87 1. PART 2
TIME OF ELUTION-DEPENDENT ELECTROPHORETIC PATTERNS OF ALTERNARIA (ALT) GLYCOPROTEINS. c Barnes PhD. F Pachcco MS. J Portnov MD. Kansas City, Missouri. Many factors contribute to the content of ALT extracts including the kinetics of glycoprotein release during extraction. To qualitatively and quantitatively demonstrate the differences between quickly and slowly &ted materials, the following study was pcrformcd. Four strains of ALT were grown for 4 weeks in defined medium under identical conditions. The fungal mats were air-dried for 24 hours, powdered with mortar and pestle, and extracted at 4°C with stirring in distilled water (DW). During extraction, all of the supcrnate was removed at 1, 3. 6, and 24 hours and was replaced with an equal volume of DW. Extraction of undissolved materials then continued during the next time interval. All preparations were dialysed YS50 mM NH4HC03 and lyophilized. Reconstitution was in DW at lOmg/ml (w/v). Protein concentrations were measuredby Lowry, and carbohydrate by the phenol-sulfuric acid method. Preparations were subjected to SDS-PAGE and analylical agarosc IEF. Western blots of the SDS-PAGE were probed for IgE binding with pooled scra from ALT-sensitive patients. Extrachblc dry weights decreaxd with each iltnc ii:icrval while carbohydrate to protein ratio increased. SDS-PAGE rcvcalcd similar bands bciwccn strains at all times, most pron~incntly at 1’7.40. and 70kD. The 17kD band appears in 3 strains at 1, 3. and G hours. but not at 24 hours. The 40 and 70kD bands wcrc notable only al 6 and 24 hours. On IEF. a band at pI 3.5 which corrclatcs with a 70kD plycoprotcin previously isolntcd from ALT by a monoclonal antibody. was mom prominent at 24 hours. Significant IgE-rccognizcd bands were seen at 35. 50.60, and 135kD. The 35kD band was primxily dctcctcd at 1 hour. The 50 and 60 kD ones were seen at 24hrs in 2 strains and didn’t vary with time in the other 2. The 135kD band was only seen at 24hrs in 2 strains. Minor bands were obscrvcd at 17 and 70kD and wcrc also time dcpcndcnt. Release of individual glycoproteins from ALT occurs at dilfcrcnt rates during clution. Several of these are uniquely present in prcparations eluted for specific time intervals. Therefore, the elution time must be optimized if production of preparations containing any particular combination of glycoprotcins is desired.
186 EFFECTS OF AIR POLLUTANTS ON HAYFEVER SYMPTOM SEVERlTY G. Gunner. M.Sc. Greta k Rilev. BSc., Penny Fitzharris. M.D. and M.R. &hmore.Ph.D., London, U.K. We investigated the influence of ambient air pollutants on the severity of hayfever symptoms in a group of 76 patients who lived and worked in central London during the 1990U.K. grasspollen smscm. Symptom scores and medication use were recorded daily for the entire seasonand data for NO2, NO, 03 and SO2 concentrations obtained from London monitoring sites. Daily central London pollen counts were also obtained. After fitting a model basedon pollen counts the extent to which the residual variation in daily symptomscould be explained hy air pollutants was examined. Although the daily pollen counts show4 a significant correlation with symptomsscore, a modified pollen count, Pmod, which allowed a lag effect from previous days’ exposure significantly improved the tit to the data. With the addition of the cumulative pollen count, Pcum,60% of the variation in mean daily symptom score (S) could be explained by a model of the form S = aI + a2lnPmod + a3lnPcum. The addition of NO2 concentrations to the model further reduced the residual variation and significantly improved the fit to the data. Other pollutants and climatic variables showed no significant effects. These results suggestthat a relatively simple model provides a good fit to the observed symptom data and, furthermore, that ambient NO2 may contribute significantly to hayfever symptom severity in London.
185
187
CROSS RFACTISYRINGA VULGARIS L. (SV),AND FRAXINUS EXCELSI;IR L. (FE): ' 4. Wahl PhD, L J. fEoig BA, L M. Wimnel BA, " A. Caneaero MD ' D-2057 Reinbek (FRG). L Barcelone. ti Sevilia (Soain) Sensibilisation-to OE-pollen~;s v&-y common in the mediterranean area. SV and FE belong to the same family as OE. We investigated the cross reactivity between the three species. The sera of 12 OE-pollen allergic patients were investigated with different techniques. Skin tests were positive with the three extracts. The strongest reaction could be seen with OE. Using allergen disks, loaded with the three pollen extracts, positive RAST-classes (R-cl) could be found. The highest could be measured with OE-allergen disks (mean R-cl 3.6). With FE and SV disks RAST-class 2.7 (mean) was measured. Using the poolserum (N=12), the three kinds of allergen disks and the three extracts (vice versa), cross reactivity of different degrees could be found by RAST-inhibition. IEF and SDS-PAGE of all extracts showed complex protein patterns. IEF determined proteins were in an IP-range between 3.7 and 5.4. By SDS-PAGE most of them could be detected in a molecular weight range between 14.4 and 94.0 kD. The immunoprint showed most of the comnon allergens in an Ip-range between 4.5 and 5.4 and westernblots showed most of the common allergens with molecular weights between 14.4 and 20.0 kD. By these blotting techniques also species-specific allergens could be detected in the three extracts. Our results show a high degree of cross reactivity between OE, FE and SV. This should be taken into account if all three species are present in the local flora.
188
mMUNOCHEMICALANALYSIS
OF BAHIA GBBss cr. w. L. TNdeau. B.A. (MOD). E. F-s Ph.D.. D. K. Ledford. M.D.. G. A. Bucholtz. M.D. and R. F. Lockev M.D.& Tampa, FL. Sensitization to Bahia grass pollen (BGP). a common United States, has been grass in the Southern documented by skin testing, RAST and nasal challange. The allergenic composition of BGP was studied by preparative scale IEF. SDS-PAGE, IEF electrophoresis. RAST and immunoblot. Twenty grams of BGP were in ammonium defatted, extracted (1 : 20 w/v) bicarbonate, dialyzed and centrifuged. Sixty ml of the supemantant (12’3 mg protein) were loaded into a with 2% ampholyte, pH 3.5-10, and Rotofor@ electrophoresed for 6 hours at 12 W. Twenty fractions were harvested. Coomassie blue staining of the IEF gel showed bands in the pH range of 4.0-9.1. RAST to the individual fractions using 6 sera demonstrated consistent IgE binding in the pH range of 4.65 to 10. Higher IgE binding was detected to 7 fractions in the pH range of 4.5-6.5 and IO 6 fractions in the pH range of 7.3-10. Immunoblots revealed 1 major IgE binding band in the acidic fractions (AF) at 31 kD and 2 bands in the basic fractions (BF) at 33 kD and 14 kD. Pooled AF (78.8 mg) and BF $6.8 mg) were electrophoresed and examined by RAST. IgE sparately in the Rotofor binding to AF remained high in the pH range 4.5-6.9 and to BF in the pH range 7.7-9.5. Fractions in these pH ranges were pooled, dialyzed, lyophylized and chromatographed on a Sephadex G-50 column. Both AF and BF eluted in two peaks. BGP has three major allergens with molecular weights of 31 kD, 33 kD and 14 kD which occur in the pH ranges of 4.5-6.5 and 7.3-10.