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169 Comparison of proteins from two strains of alternaria (ALT) using hydrophobic interaction chromatography
VOLUME 87 NUMBER 1. PART 2
Abstracts
169 COMPARISONOF PROTEINS 6ROM TWO STRAINS OF ALTERNARIA (ALT) USING HYDROPHOBIC INTERACTION CHROMATOGRAPHY. J’ Ballam BS. FPachcco MS. C.B,w PhD. J.Portnov MD, KansasCily, Missouri. Elecbophoretic separation and quantification of glycoproteins in extracted ALT preparations is complicated by the large number of individual componentspresent. Hydrophobic interaction chromatography (HIC) using phenyl spcharoscis a proccdurc for separating glycoproteins according to their hydrophobicity and carbohydrate content. Proteins thus separatedcan lhen be more easily identified and quantified by traditional electrophoretic methods.To compare the protein componentsof two ALT strains, the following study was performed. Two strains of ALT were grown for 4 weeks in detincd medium under identical conditions. The fungal mats were air dried for 24 hours, powdered with a mortar and pestle, and extracted at 4°C for 48 hours. Extracts were then diilyzcd against 50mM NH4HC03 and lyophilized. The preparations wcrc reconstituted in with OSM Tris, pII 7.5 containing 4 M NaCI, and applied to a phenyl scphzuose column. Next, the column was eluted stepwise with buffer containing 4. 2, and 1 M NaCl and finally wilh distilled water. The eluted material was dialyzed against NILHCO3 and lyophilizcd. Protein content was determined by the Lowry method and carbohydrate was by the phenol sulfuric acid mclhod. SDS-PAGE w&qpcrformcd by Ihc method of Lamclli.
171
QET_EXJ-nJLF &~Ni~B00IES AGAINST _-. I oG :[!-rERMClACTINOMYCES VULGARIS ANTIGENS -.u HYPERSENSXVITY PNEUMONITIS PATIENTS ---....---A Sadhna Jain Rita -.-LA%.&--Kumar PhD DeC?pak: -----.----e,MSL,p !-ha,. J3._S_ro Sharad V. Ganqal, f’h .D. ,. Delhi.Indla The bodies
presence af (Abs! against
of allergen activity
in ALT extracts has been
Measurement of histamine rclcase from lcukocytcs of 3 ALT-
sensitive patients revealed no rclcase in 1. and poor corrckuion bctwccn the other 2 for the diflcrenttimes. Each rcspondcd most strongly to diflcrent preparations. Multiple allergens are released from ALT at diffcrcnt limes during ant&n elution. Individuals seem to react IO materials that
may be uniquely present in preparations eluted for diffcrcnt time intervals. This suggeststhe possiblity of producing qualitatively different extracts of the same mold that are most appropriate for a particulx individualmerely by varying the time of elution.
anti-
for
its ablllty to hxnsl ttle serum 1gG. Out of the 40 patients of HP studied, 27 patient3 showed the presence of I tgG Abs. The patlent 11rlss on Dl.D also of t h e 5 fratrtlons fr‘?ctlan 1v and
not 0J’““‘4 preclpitln t1tres. out gave ELI% obtained on FPLC, v were found to be
Ill~llly nnt.l~Jenlr by El-ISA lnt,lbltl~,n test u5 I 174 PCJOiCd patients sera. The total number of antlgenic iamponents of T.vulqarir with CIE revealed presence of 24-25 bands in crude e:
11,
~.VLll~~drl~ test,
f'o r
positive means of
Variability
1gG
(HP) patierlts in Ind1a. T.vulqaris in 5erd of patients were detected by double immunodiffusio” (DID), ELISA and Imrr,unoblot,. Antigens were fractionated wc75 studled on FPLC and each fraction
species are noted especially in the 20KD region of the 2M fraction and the 30KD region of the 1M and DW fractions. HIC with phcnyl scpharose is a good method for separating ALT proteins in extracts. Using lhis technique many inter-strain similaritics and dlffcrcnccs are noted.
attributed to many factors including the kin&s of allergen release during extraction. To dctcrminc whcthcr glycoproteins that arc quickly clutcd will advcrscly cffcct allcrgcn activity that may bc released subsequently,the following study was performed. 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 wilh stirring in distilled water (DW). During extraction, all of the supemale was removed at 1, 3, 6. and 24 hours and replaced with an equal volume of DW. Extraction of the undissolved material then conlinued during the next time interval. All preparations were dialyscd \‘s 50 mM NH4HC03 and lyophilizcd. Reconstitution was in DW at IOmg/ml (w/v). Biologic activity was measuredby IgG and IgE ELISA inhibition using pooled serum and by leukocyte histuminc rclcase of 3 patients who are skin-reactive to ALT. Extracts did not significantly differ when lhcir potency was measuredby IgG ELISA inhibition. For IgE, maximum inhibition was seen in a mixture of all extracts. Thcrc were significant differences in IgE inhibition at different extraction intervals for individual strains. These time-dcpcndcnt variations wcrc not consislcnt between different strains. The only extraction lime that consistently yielded high activity in all strains was at 6 hours.
tc
TJermoactinamyces. in hyper-
sensit,ivity pneumonitis has not bren demonstrated The speclflc Abs against
only one or two wash fractions. Protein similarilies bctwecn the two
(AL-Q PREPARATIONS TIMEINTERVALS.JPortnov EXTRACTEDFORDDFFERENT MD. F Pachcco MS. C mcs PhD. Kansas City, Missouri.
specif
fT.vulqaris)
.v_u.Lsz22
Signil’nxnu varialions of prolcin and cartx9lydratc co111cnl bctwccn washes were observed for bolh strains with the CHO/PRO ralio being highest in the 4 Molar wash. Elcclrophorctic analysis of scparatcd fractions dcmonstratcs clear diffcrcnccs in protein cornponcnts hctwcen Ihe salt washes with some proteins appearing in
170 ALLERGEN ACTIVITY OFALTERh’ARIA
181
172
India. specific
screening
negative
results.
suggest
the
T..vxLqaa;n
Although IgG of
I5 d pcit1ent5
presence
in ELI% suns1
t-esuiis sh0uirl tre confirmed immunoblot analysis.
H.P. of t1ve sera,
by
A MONOCLONAL ANTIBODY AGAINST THE 68KD ALLERGEN OF PENICILLIUJ NOTATUM. H.D.Shen,.MS, S.R.Wang, MD, Ph.D., S.H, Taiwan, R.O.C. Han, MD, Ph.D., Taipei, Penicillium -notatum has been considered to be a causative agent of extrinsic bronchial asthma. However,little was known about the allergenic composition crossof P. -notatum and the allergenic and reactivity among the Penicillium present fungi species. In the other was 68-kD component study, the major allergen of identified to be the P. notatum by SDS-PAGE and immunoblot. this allergen has ii MoAb (~40) against Analyzed by twoalso been generated. electrophoresis and dimensional gel ~40 reacted with 68-kD immunoblotting, components of p. notatum with p1 values P40 showed positive ELISA from 6.0- 6.1. but activity to Aspergillus fumigatus to components of i0 negative activity other air-borne fungi. By SDS-PAGE and ~40 also showed positive reimmunoblot, 67-kD components of P. the activity to frequentans and P. roseopurpureum butnot to components of the 8 other species of ____. Penicillium which have been reported with asthma. Furtherto be associated imni;noP40 also showed positive more, blot activity to the 67-kD component of A. fumigatus which was identified to be .n allergen also by immunoblotting. The allergenic cross-reactivities described diagnostic the should be considered in clinical allergy.
Abstracts
169 COMPARISONOF PROTEINS 6ROM TWO STRAINS OF ALTERNARIA (ALT) USING HYDROPHOBIC INTERACTION CHROMATOGRAPHY. J’ Ballam BS. FPachcco MS. C.B,w PhD. J.Portnov MD, KansasCily, Missouri. Elecbophoretic separation and quantification of glycoproteins in extracted ALT preparations is complicated by the large number of individual componentspresent. Hydrophobic interaction chromatography (HIC) using phenyl spcharoscis a proccdurc for separating glycoproteins according to their hydrophobicity and carbohydrate content. Proteins thus separatedcan lhen be more easily identified and quantified by traditional electrophoretic methods.To compare the protein componentsof two ALT strains, the following study was performed. Two strains of ALT were grown for 4 weeks in detincd medium under identical conditions. The fungal mats were air dried for 24 hours, powdered with a mortar and pestle, and extracted at 4°C for 48 hours. Extracts were then diilyzcd against 50mM NH4HC03 and lyophilized. The preparations wcrc reconstituted in with OSM Tris, pII 7.5 containing 4 M NaCI, and applied to a phenyl scphzuose column. Next, the column was eluted stepwise with buffer containing 4. 2, and 1 M NaCl and finally wilh distilled water. The eluted material was dialyzed against NILHCO3 and lyophilizcd. Protein content was determined by the Lowry method and carbohydrate was by the phenol sulfuric acid mclhod. SDS-PAGE w&qpcrformcd by Ihc method of Lamclli.
171
QET_EXJ-nJLF &~Ni~B00IES AGAINST _-. I oG :[!-rERMClACTINOMYCES VULGARIS ANTIGENS -.u HYPERSENSXVITY PNEUMONITIS PATIENTS ---....---A Sadhna Jain Rita -.-LA%.&--Kumar PhD DeC?pak: -----.----e,MSL,p !-ha,. J3._S_ro Sharad V. Ganqal, f’h .D. ,. Delhi.Indla The bodies
presence af (Abs! against
of allergen activity
in ALT extracts has been
Measurement of histamine rclcase from lcukocytcs of 3 ALT-
sensitive patients revealed no rclcase in 1. and poor corrckuion bctwccn the other 2 for the diflcrenttimes. Each rcspondcd most strongly to diflcrent preparations. Multiple allergens are released from ALT at diffcrcnt limes during ant&n elution. Individuals seem to react IO materials that
may be uniquely present in preparations eluted for diffcrcnt time intervals. This suggeststhe possiblity of producing qualitatively different extracts of the same mold that are most appropriate for a particulx individualmerely by varying the time of elution.
anti-
for
its ablllty to hxnsl ttle serum 1gG. Out of the 40 patients of HP studied, 27 patient3 showed the presence of I tgG Abs. The patlent 11rlss on Dl.D also of t h e 5 fratrtlons fr‘?ctlan 1v and
not 0J’““‘4 preclpitln t1tres. out gave ELI% obtained on FPLC, v were found to be
Ill~llly nnt.l~Jenlr by El-ISA lnt,lbltl~,n test u5 I 174 PCJOiCd patients sera. The total number of antlgenic iamponents of T.vulqarir with CIE revealed presence of 24-25 bands in crude e:
11,
~.VLll~~drl~ test,
f'o r
positive means of
Variability
1gG
(HP) patierlts in Ind1a. T.vulqaris in 5erd of patients were detected by double immunodiffusio” (DID), ELISA and Imrr,unoblot,. Antigens were fractionated wc75 studled on FPLC and each fraction
species are noted especially in the 20KD region of the 2M fraction and the 30KD region of the 1M and DW fractions. HIC with phcnyl scpharose is a good method for separating ALT proteins in extracts. Using lhis technique many inter-strain similaritics and dlffcrcnccs are noted.
attributed to many factors including the kin&s of allergen release during extraction. To dctcrminc whcthcr glycoproteins that arc quickly clutcd will advcrscly cffcct allcrgcn activity that may bc released subsequently,the following study was performed. 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 wilh stirring in distilled water (DW). During extraction, all of the supemale was removed at 1, 3, 6. and 24 hours and replaced with an equal volume of DW. Extraction of the undissolved material then conlinued during the next time interval. All preparations were dialyscd \‘s 50 mM NH4HC03 and lyophilizcd. Reconstitution was in DW at IOmg/ml (w/v). Biologic activity was measuredby IgG and IgE ELISA inhibition using pooled serum and by leukocyte histuminc rclcase of 3 patients who are skin-reactive to ALT. Extracts did not significantly differ when lhcir potency was measuredby IgG ELISA inhibition. For IgE, maximum inhibition was seen in a mixture of all extracts. Thcrc were significant differences in IgE inhibition at different extraction intervals for individual strains. These time-dcpcndcnt variations wcrc not consislcnt between different strains. The only extraction lime that consistently yielded high activity in all strains was at 6 hours.
tc
TJermoactinamyces. in hyper-
sensit,ivity pneumonitis has not bren demonstrated The speclflc Abs against
only one or two wash fractions. Protein similarilies bctwecn the two
(AL-Q PREPARATIONS TIMEINTERVALS.JPortnov EXTRACTEDFORDDFFERENT MD. F Pachcco MS. C mcs PhD. Kansas City, Missouri.
specif
fT.vulqaris)
.v_u.Lsz22
Signil’nxnu varialions of prolcin and cartx9lydratc co111cnl bctwccn washes were observed for bolh strains with the CHO/PRO ralio being highest in the 4 Molar wash. Elcclrophorctic analysis of scparatcd fractions dcmonstratcs clear diffcrcnccs in protein cornponcnts hctwcen Ihe salt washes with some proteins appearing in
170 ALLERGEN ACTIVITY OFALTERh’ARIA
181
172
India. specific
screening
negative
results.
suggest
the
T..vxLqaa;n
Although IgG of
I5 d pcit1ent5
presence
in ELI% suns1
t-esuiis sh0uirl tre confirmed immunoblot analysis.
H.P. of t1ve sera,
by
A MONOCLONAL ANTIBODY AGAINST THE 68KD ALLERGEN OF PENICILLIUJ NOTATUM. H.D.Shen,.MS, S.R.Wang, MD, Ph.D., S.H, Taiwan, R.O.C. Han, MD, Ph.D., Taipei, Penicillium -notatum has been considered to be a causative agent of extrinsic bronchial asthma. However,little was known about the allergenic composition crossof P. -notatum and the allergenic and reactivity among the Penicillium present fungi species. In the other was 68-kD component study, the major allergen of identified to be the P. notatum by SDS-PAGE and immunoblot. this allergen has ii MoAb (~40) against Analyzed by twoalso been generated. electrophoresis and dimensional gel ~40 reacted with 68-kD immunoblotting, components of p. notatum with p1 values P40 showed positive ELISA from 6.0- 6.1. but activity to Aspergillus fumigatus to components of i0 negative activity other air-borne fungi. By SDS-PAGE and ~40 also showed positive reimmunoblot, 67-kD components of P. the activity to frequentans and P. roseopurpureum butnot to components of the 8 other species of ____. Penicillium which have been reported with asthma. Furtherto be associated imni;noP40 also showed positive more, blot activity to the 67-kD component of A. fumigatus which was identified to be .n allergen also by immunoblotting. The allergenic cross-reactivities described diagnostic the should be considered in clinical allergy.