Immunochemical characterization of edible bird’s nest allergens

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Presentation transcript:

Immunochemical characterization of edible bird’s nest allergens Denise Li Meng Goh, MMed (Paeds), MRCPa, Kaw Yan Chua, PhDa, Fook Tim Chew, PhDa, Rosa Cynthia Mui Yee Liang, BSc, Teck Keong Seow, PhDb, Ke Li Ou, PhDb, Fong Cheng Yi, BSc (Hons)a, Bee Wah Lee, MDa Journal of Allergy and Clinical Immunology Volume 107, Issue 6, Pages 1082-1088 (June 2001) DOI: 10.1067/mai.2001.114342 Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 1 A , SDS (7.5%) protein profiles. Lane 1 , Fresh BN from Sarawak (20 μg/lane); lane 2 , commercially available BN from Sarawak BN (20 μg/lane); lane 3 , BN from Indonesia (20 μg/lane); and lane 4 , BN from Thailand (20 μg/lane). B , IgE immunoblot done with fresh unprocessed BN from Sarawak with allergic sera (patient 1, lane 1 ) and negative control (lane 2) . MW , Molecular weight marker. Journal of Allergy and Clinical Immunology 2001 107, 1082-1088DOI: (10.1067/mai.2001.114342) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 2 FAST inhibition curves. Solid phase is defined as commercially available processed BN from Sarawak. Liquid phase is defined as pooled positive sera preabsorbed with commercially available processed BN from Sarawak (circles) , Thailand (triangles) , and Indonesia (squares) or BSA (asterisks , negative control). Levels of BN-specific IgE after preabsorption were determined by means of FAST assay. Journal of Allergy and Clinical Immunology 2001 107, 1082-1088DOI: (10.1067/mai.2001.114342) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 3 IgE immunoblots with one-dimensional protein profile of fresh BN from Sarawak. Thirty micrograms of protein was loaded to each lane of a 7.5% SDS-PAGE gel separated by means of vertical electrophoresis and electroblotted to nitrocellulose. Blots were blocked with 3% nonfat milk in 0.05% PBST for 1 hour, incubated with sera (2× dilution with PBST + 1% nonfat milk) for 18 hours at 4°C, and developed with ECL-plus reagent (Amersham). Lanes 1 to 12 , Immunoblots done by using positive sera from 12 allergic patients; -ve , Negative control; MW , molecular weight marker. Journal of Allergy and Clinical Immunology 2001 107, 1082-1088DOI: (10.1067/mai.2001.114342) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 4 IgE immunoblots by using 2-dimensional protein profile of fresh BN from Sarawak. Protein load was approximately 100 μg of protein per gel. The first dimension of IEF was performed on 13-cm pH 4-7 IPG strips (Amersham Pharmacia). Second dimension separation was carried out by using 13% SDS-PAGE. The separated proteins were electroblotted to nitrocellulose, blocked with 3% nonfat milk in 0.05% PBST for 1 hour, incubated with sera (5× dilution in PBST + 1% nonfat milk) for 18 hours at 4°C, and developed with ECL-plus reagent (Amersham). A , Immunoblot done with serum from patient 1 (see Fig 2). B , Immunoblot done with negative control. Journal of Allergy and Clinical Immunology 2001 107, 1082-1088DOI: (10.1067/mai.2001.114342) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 5 Effect of periodate treatment of BN on IgE immunoblots. Single nitrocellulose strips with electroblotted BN proteins were oxidized with 0.1 mol/L sodium periodate (100 mmol/L periodic acid in 10 mmol/L sodium acetate, pH 5.0) or sodium acetate buffer alone (control) for 1 hour at room temperature. The blots were subsequently blocked, incubated with sera (5× dilution in PBST plus 1% nonfat milk) for 18 hours at 4°C, and developed with ECL-plus reagent (Amersham). The IgE-binding patterns are shown. Lane 1 , Untreated blot with positive sera; lane 2 , untreated blot with negative control; lane 3 , treated blot with positive sera; and lane 4 , treated blot with negative control. Journal of Allergy and Clinical Immunology 2001 107, 1082-1088DOI: (10.1067/mai.2001.114342) Copyright © 2001 Mosby, Inc. Terms and Conditions

Fig. 6 Two-dimensional SDS-PAGE gel showing protein profiles of fresh BN from Sarawak and the effect of heat treatment. A , Fresh unboiled BN from Sarawak (187 μg of protein per gel); B , fresh BN from Sarawak boiled for 10 minutes (148 μg of protein per gel); and C , fresh BN from Sarawak boiled 30 minutes (166 μg of protein per gel). Journal of Allergy and Clinical Immunology 2001 107, 1082-1088DOI: (10.1067/mai.2001.114342) Copyright © 2001 Mosby, Inc. Terms and Conditions