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972 SDS-polyacrylamide gel electrophoressi (SDS-PAGE) analysis of allergenic extracts
Abstracts
J ALLERGY CLIN IMMUNOL VOLUME 105. NUMBER 1. PART 2
970
Cross Reactivity of Northern Grass Allergy Skin Tests M Valyasevi. S F/.vnn, PS Kelkac JTLi Allergy and Internal Medicine, Mayo Clinic, Rochester, MN BACKGROUND: There is significant cross-allergenicity among the northern grasses [Kentucky Blue (K): Orchard (0); Timothy (T)]. The clinical significance and extent of cross-allergenicity is not fully known. OBJECTIVE: To determine the appropriate number of grass pollen allergy skin tests in patients with suspected grass allergy. METHOD: This retrospective study used a computerized database at the Mayo Clinic to identify patients who had positive skin tests to K. 0, and T. Altogether K, 0, and T skin prick tests were performed on 4,427 patients from January I, 1997. to March 3 I, 1999. Nine hundred and nineteen patients had positive skin tests. RESULTS:
NORTHERN
GRASS
SKINTESTSRESULTS
ALLERGY
SKIN
PATIENTS
TESTS (NUMBER)
PATIENTS
K+. O+. T+
717
78
K+, O+. T-
44
4.8
K+. 0-. T+
24
2.6
K-. O+. T+ K+ o+ T+ TOTAL
29
(%I
3.2
47
5.1
39
4.2
19
2.1
919
loo
Significant percentages (22%) of grass-sensitive patients were sensitized to only I or 2 of the major grasses. There was no difference in the pattern of skin tests responses to K. 0, or T. CONCLUSION: Although the mojority (78%) of patients was sensitized to all 3 major grasses, a significant minority (22%) was sensitized to only I or 2 grasses. We recommend including all 3 major northern grasses in a skin test panel. 971
Determination Extracts Using
of Der p 2 in Dermatophagoides pteronyssinus rDer p 2 and Scanning Densitometry E Ferndnde:-C&as, ML Gonzdlez Romano, MT Gallego C.B.F. LETI. S.A.. Research Laboratories, Madrid, Spain We have previously demonstrated that scanning densitometry with purified allergens can be used to determine major allergen content of allergen extracts (J Immunol Methods 1999: 223: 17-26). The objectives of this study were: I) to determine Der p 2 content in D. pteronyssinus extracts using rDer p 2 and scanning densitometry and 2) to compare the results with those obtained with monoclonal antibodies. A standard curve with serial dilutions of r Der p 2 starting at 2 pg per lane and I4 D. pteronyssinus extracts were run under reducing conditions in each SDS-PAGE gels. The gels were stained and scanned with the SHARP Film Scanning Unit (Model JX-3F6) and a SHARP JX-330 Color scanner. Lanes were analyzed using the ImageMaster software from Pharmacia Biotech.
S329
The experiment was repeated twice to evaluate the reproducibility of the method. Extracts were also analyzed for Der p 2 content using monoclonal antibodies (Indoor Biotechnologies Ltd.). The method was highly consistent (r=O.94; p=O.OOOl). Der p 2 levels ranged from I to I5 pg/mg using ELISA and from 2 to I I pg/mg of lyophilized material using scanning densitometry. There was a good correlation between the results obtained through scanning densitometry and monoclonal antibodies, r=O.72; p=O.O04. Scanning densitometry is a valid and reproducible alternative to monoclonal antibodies to measure Der p 2 in mite extracts. 972
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analyisis of Allergenic Extracts GA Plunkett, LD Baldwin, RE Baker; LM Featherstone Hollister-Stier Laboratories LLC, Spokane, WA, USA Most allergenic extracts sold in the USA are non-standardized. The only standardized extracts are house dust mites and some grass extracts (potency determined by IgE binding ELISA inhibition), cat and short ragweed extracts (potency determined by the amount of a major allergen), and some insect venom extracts (potency determined by enzyme levels and total protein). Activity of non-standardized extracts relies on the Protein Nitrogen Unit (PNU) which does not correlate well with other activity assays such as IgE ELISA inhibition. The purpose of this study is to evaluate SDSPAGE as a technique to characterize non-standardized allergenic extracts based upon the visualized protein profile. This technique identifies proteins in extracts and separates them in a gel by their molecular weight (MW). The method was validated by assaying one lot of a non-standardized Russian thistle extract 90 times. The study showed that the method was reproducible by: I) repeatedly producing identical gel patterns, and 2) the MW of selected proteins, as determined using gel analysis software, had a variability of less than 2% (percentage CV)(GA Plunkett, et al., JACI 103: S30, 1999). Regular production batches of allergenic extracts were analyzed by SDS-PAGE. Evaluation of over 700 lots representing 170 different allergen extract types showed that most produced patterns with a unique protein array. Multiple lots of the same extract gave patterns that were similar for most allergen types, even when different lots of source material were used. For some extract types, aqueous and glycerinated extractions produced different gel protein patterns, which may be a function of the extractability of particular proteins in the different extraction fluids. This was sometimes due to missing protein bands. In many cases, prominent proteins at MWs identidied in literature references as major allergens were evident in the gels. In conclusion, SDS-PAGE can characterize allergen extracts by producing a pattern of the predominant proteins. The technique can be used to assess the source material and manufacturing conditions of extracts.
J ALLERGY CLIN IMMUNOL VOLUME 105. NUMBER 1. PART 2
970
Cross Reactivity of Northern Grass Allergy Skin Tests M Valyasevi. S F/.vnn, PS Kelkac JTLi Allergy and Internal Medicine, Mayo Clinic, Rochester, MN BACKGROUND: There is significant cross-allergenicity among the northern grasses [Kentucky Blue (K): Orchard (0); Timothy (T)]. The clinical significance and extent of cross-allergenicity is not fully known. OBJECTIVE: To determine the appropriate number of grass pollen allergy skin tests in patients with suspected grass allergy. METHOD: This retrospective study used a computerized database at the Mayo Clinic to identify patients who had positive skin tests to K. 0, and T. Altogether K, 0, and T skin prick tests were performed on 4,427 patients from January I, 1997. to March 3 I, 1999. Nine hundred and nineteen patients had positive skin tests. RESULTS:
NORTHERN
GRASS
SKINTESTSRESULTS
ALLERGY
SKIN
PATIENTS
TESTS (NUMBER)
PATIENTS
K+. O+. T+
717
78
K+, O+. T-
44
4.8
K+. 0-. T+
24
2.6
K-. O+. T+ K+ o+ T+ TOTAL
29
(%I
3.2
47
5.1
39
4.2
19
2.1
919
loo
Significant percentages (22%) of grass-sensitive patients were sensitized to only I or 2 of the major grasses. There was no difference in the pattern of skin tests responses to K. 0, or T. CONCLUSION: Although the mojority (78%) of patients was sensitized to all 3 major grasses, a significant minority (22%) was sensitized to only I or 2 grasses. We recommend including all 3 major northern grasses in a skin test panel. 971
Determination Extracts Using
of Der p 2 in Dermatophagoides pteronyssinus rDer p 2 and Scanning Densitometry E Ferndnde:-C&as, ML Gonzdlez Romano, MT Gallego C.B.F. LETI. S.A.. Research Laboratories, Madrid, Spain We have previously demonstrated that scanning densitometry with purified allergens can be used to determine major allergen content of allergen extracts (J Immunol Methods 1999: 223: 17-26). The objectives of this study were: I) to determine Der p 2 content in D. pteronyssinus extracts using rDer p 2 and scanning densitometry and 2) to compare the results with those obtained with monoclonal antibodies. A standard curve with serial dilutions of r Der p 2 starting at 2 pg per lane and I4 D. pteronyssinus extracts were run under reducing conditions in each SDS-PAGE gels. The gels were stained and scanned with the SHARP Film Scanning Unit (Model JX-3F6) and a SHARP JX-330 Color scanner. Lanes were analyzed using the ImageMaster software from Pharmacia Biotech.
S329
The experiment was repeated twice to evaluate the reproducibility of the method. Extracts were also analyzed for Der p 2 content using monoclonal antibodies (Indoor Biotechnologies Ltd.). The method was highly consistent (r=O.94; p=O.OOOl). Der p 2 levels ranged from I to I5 pg/mg using ELISA and from 2 to I I pg/mg of lyophilized material using scanning densitometry. There was a good correlation between the results obtained through scanning densitometry and monoclonal antibodies, r=O.72; p=O.O04. Scanning densitometry is a valid and reproducible alternative to monoclonal antibodies to measure Der p 2 in mite extracts. 972
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analyisis of Allergenic Extracts GA Plunkett, LD Baldwin, RE Baker; LM Featherstone Hollister-Stier Laboratories LLC, Spokane, WA, USA Most allergenic extracts sold in the USA are non-standardized. The only standardized extracts are house dust mites and some grass extracts (potency determined by IgE binding ELISA inhibition), cat and short ragweed extracts (potency determined by the amount of a major allergen), and some insect venom extracts (potency determined by enzyme levels and total protein). Activity of non-standardized extracts relies on the Protein Nitrogen Unit (PNU) which does not correlate well with other activity assays such as IgE ELISA inhibition. The purpose of this study is to evaluate SDSPAGE as a technique to characterize non-standardized allergenic extracts based upon the visualized protein profile. This technique identifies proteins in extracts and separates them in a gel by their molecular weight (MW). The method was validated by assaying one lot of a non-standardized Russian thistle extract 90 times. The study showed that the method was reproducible by: I) repeatedly producing identical gel patterns, and 2) the MW of selected proteins, as determined using gel analysis software, had a variability of less than 2% (percentage CV)(GA Plunkett, et al., JACI 103: S30, 1999). Regular production batches of allergenic extracts were analyzed by SDS-PAGE. Evaluation of over 700 lots representing 170 different allergen extract types showed that most produced patterns with a unique protein array. Multiple lots of the same extract gave patterns that were similar for most allergen types, even when different lots of source material were used. For some extract types, aqueous and glycerinated extractions produced different gel protein patterns, which may be a function of the extractability of particular proteins in the different extraction fluids. This was sometimes due to missing protein bands. In many cases, prominent proteins at MWs identidied in literature references as major allergens were evident in the gels. In conclusion, SDS-PAGE can characterize allergen extracts by producing a pattern of the predominant proteins. The technique can be used to assess the source material and manufacturing conditions of extracts.