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- Marknell DeWitt, Åsa, 1966-
(author)
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Use of Recombinant Allergens for Component-Resolved Diagnostics (CRD) in IgE-Mediated Allergy
- 2007
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Doctoral thesis (other academic/artistic)abstract
- Immunoglobulin E (IgE)-mediated allergy occurs when our immune system causes a reaction to otherwise harmless substances (allergens). Allergens are predominantly proteins present in biological materials such as pollens, mites, animal epithelia, moulds and foods. In vitro tests for specific IgE antibodies usually employ an allergen source extract as an antibody capturing reagent. The proportion of allergenic molecules in these biochemically complex extracts may vary.Recombinant allergens may be obtained in large quantities with biotechnological techniques. These proteins can be characterized biochemically and immunologically, resulting in tests with minimal batch-to-batch variation. This thesis describes different uses of recombinant allergens in component-resolved diagnostics (CRD).In CRD, single allergenic proteins are used to establish a sensitization profile of the patient. Two timothy grass (Phleum pratense) pollen allergens, Phl p 11 and Phl p 4, were cloned and expressed as recombinant proteins. They were subsequently characterized and can, for example, be used in a panel for grass pollen CRD.Single allergens may be useful as diagnostic markers for allergic sensitization. This phenomenon was studied using tropomyosin, a major allergen from the shrimp Penaeus aztecus (Pen a 1). The characteristics of the recombinant and natural proteins were compared. The recombinant tropomyosin was then extensively tested using specific competition for IgE binding against extracts of other crustacean species, house dust mite and cockroach.In cases when an important allergen is missing or underrepresented in a natural extract, the corresponding recombinant allergen may be added to the extract as a spiking reagent. Previous studies have shown that latex extracts for diagnostic testing may lack the allergen Hev b 5. Recombinant Hev b 5 was expressed from a synthetic gene construct, incorporating several adaptations to enable efficient large scale production of the recombinant protein, to be used as a spiking reagent.
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- Movérare, Robert, et al.
(author)
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Human Allergen-Specific IgG Subclass Antibodies Measured Using ImmunoCAP Technology
- 2017
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In: International Archives of Allergy and Immunology. - : KARGER. - 1018-2438 .- 1423-0097. ; 172:1, s. 1-10
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Journal article (peer-reviewed)abstract
- Background: Knowledge of human IgG subclass antibody responses to various allergens has been hampered by a lack of reliable standardized assays. The aim here was to develop quantitative immunoassays for human IgG1, IgG2, and IgG3 antibodies using ImmunoCAP (R) technology and to evaluate their application. Methods: Enzyme conjugates with isotype-specific monoclonal antibodies and calibrators composed of purified myeloma paraproteins were developed for each assay and used together with other standardized assay reagents for the Phadia (R) 100 instrument. The calibrators were adjusted to the international reference preparation IRP 67/86. The assays were characterized and used together with other standard ImmunoCAP assays to measure antibodies to various allergens in preliminary studies. Results: The new assays had limits of quantitation of 1.0 (IgG1), 4.6 (IgG2), and 0.04 mg(A)/L (IgG3), and coefficients of variation of <20%. Only some minor cross -reactivity with IgG2 was observed for the specific IgG1 assay. The specific IgG2 assay showed a bias for the allotype G2m(23) and compensation factors were used to adjust the measured concentrations accordingly. Preliminary studies indicated a strong and stable IgG4 antibody response to P-lactoglobulin in healthy individuals, a high IgG1 and even higher IgG2 antibody response to house dust mite in sensitized and nonsensitized subjects, and a mixed IgG subclass response to venom allergens in allergic patients with increasing IgG4 antibody levels during venom immunotherapy. Conclusions: The new research assays are valuable tools for immunological studies, enabling the characterization of antibody profiles using a standardized approach, and facilitating data interpretation and the comparison of results across studies.
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