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Two-Dimensional Gel Electrophoretic Analysis with Immobilized pH Gradients of Dactylis glomerata Pollen Allergens

Two-Dimensional Gel Electrophoretic Analysis with Immobilized pH Gradients of Dactylis glomerata Pollen Allergens,10.1159/000236553,International Arch

Two-Dimensional Gel Electrophoretic Analysis with Immobilized pH Gradients of Dactylis glomerata Pollen Allergens   (Citations: 9)
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We studied Orchard grass pollen allergens (Dactylis glomerata) by a new two-dimensional electrophoretic technique which is the combination of isoelectric focusing (IEF) in immobilized pH gradient (IPG) and horizontal SDS-PAGE. The IPG-Dalt (two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension) electrophoresis was made with four different but complementary pH gradients: 3–10.5, 4–9, 4–7, 6.3–10.5. We also compared the behavior of allergens in native and denaturing conditions during IEF. This technique followed by electroblotting on nitrocellulose membrane and immunodetection with an allergic patient serum was shown to increase the number of IgE-binding components up to 100 compared to one-dimensional techniques. Major allergen groups could be identified. New allergens not yet described could be characterized by their isoelectric points and their molecular masses. This highly resolving and reproducible technique, should allow an easy allergen standardization.Copyright © 1993 S. Karger AG, Basel
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    • ...Antibody-producing hybridomas were screened by a radioimmunoassay with Sepharose-coupled goat-anti-mouse IgG (GoMo17, Sanquin, Amsterdam, The Netherlands) and 125I-labeled affinity-purified Lol p 5. By using Lol p 5 instead of Phl p 5, screening was deliberately biased towards cross-reactive group 5 mAbs. Cloning was performed by repeated limiting dilution. This procedure resulted in 6 Balb/c-derived mAbs (2C8, 11D11, 13D9, 18B11, 14C2 and 19F9) and 2 B-10-derived mAbs (1G6 and 8E2).Group-5-Specific mAb-Based Sandwich ELISAA specific mAb-based sandwich ELISA was developed for quantification of group 5 allergens in grass pollen extracts. The ELISA was developed with three mAbs that were selected on the basis of their recognition profile of group 5 isoforms on immunoblot after separation of a grass pollen extract by 2D electrophoresis [...

    Erica van Oortet al. The Major Grass Pollen Group 5 Allergen from Dactylis glomerata and It...

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