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A method for quantification of allergens

An allergenic, absolute quantity technique, applied in the direction of measuring devices, markers used in chemical analysis, instruments

Inactive Publication Date: 2008-10-15
ALK ABELLO SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But there are still issues with cross-reactivity, matrix effects and food processing

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  • A method for quantification of allergens
  • A method for quantification of allergens
  • A method for quantification of allergens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0147] Identification of unique constant regions for Der f2, Der p2, Phl p1, Phl p5, and Bet v1 along with synthetic calibration standard peptides

[0148] Absolute quantification of allergens using unique constant region sequences, ie marker peptides in native Der f2, Der p2, Phl p1, Phl p5 and Bet v1, can be demonstrated using two different means. Synthesis of two calibration standard peptides for iTRAQ TM Labeling technology (Applied Biosystems) was evaluated. In addition, four calibration standard peptides were synthesized to evaluate Protein-AQUA TM (Sigma-Aldrich) and other isotope labeling techniques.

[0149] Based on the amino acid sequence alignment (Vector NTI) (Figure 1), the cleavage analysis carried out by GPMAW ( figure 2 ) and Blast database search to design allergen calibration standard peptides with corresponding to 32-48 in Der f2 and Der p2, 149-158 in Phl p1, 123-135 in Phl p 5a, Phl p 5b Species-specific sequences of the amino acid sequences 115-127 ...

Embodiment 2

[0152] Purified natural and recombinant grass, birch and mite group 2 allergens

[0153]Native Der f2 and Der p2 were purified from 100 mg of Dermatophex farinae and house dust mite extracts (ALK-Abelló, Horsholm, Denmark). Native Phl p 1 and 5 were purified from 50 mg Phleum prantense extract (ALK-Abelló) and native Bet v1 was purified from 50 mg Betulaverrucosa extract (ALK-Abelló). Purification of the molecules was performed as described (Johannessen BR et al. FEBS Lett 2005; 579: 1208-12, Aasmul-Olsen S. et al. New Horizons in Allergy Immunotherapy, edit by Sehon et al. Plenum Press. New York 1996, p.261-65, Petersen A et al. Clin Exp Allergy. 1994 Mar;24(3):250-6, Ipsen H & Lowenstein H J Allergy Clin Immunol. 1983;72(2):150-59). Recombinant Der f2 and Der p2 were expressed in the Pichia pastoris expression system and purified as described (Johannessen BR et al. FEBS Lett 2005;579:1208-12). Proteins were stored at -20°C as lyophilized aliquots.

[0154] Using one of th...

Embodiment 3

[0156] Pre-fractionation with HDM extract and protein digestion

[0157] Pre-fractionation of dust mite (Der f) and house dust mite (Der p) extracts by hydrophobic interaction chromatography. Fractionation of mite extracts was performed with a 1.0 ml HiTrap Phenyl column (GE-Healthcare, Uppsala, Sweden). Equilibrate the column with 50 mM sodium phosphate buffer (Merck, Darmstadt, Germany), pH 7.0, 1.0 M ammonium sulfate (Fluka, Buchs, Switzerland), buffered with 5 column volumes of 50 mM sodium phosphate with a decreasing linear gradient. solution (Merck), pH 7.0 to elute the bound sample. Chromatography was performed separately for each of the HDM extracts (Der f and Der p). Based on the analysis of the fractions by SDS-PAGE (Invitrogen, Carlsbad, CA, USA), the HDM proteins were divided into two major protein pools ( Figure 4 ). Der f and Der p pools containing HDM 2 allergen were dialyzed against 10 mM ammonium bicarbonate (BDH, Poole, England). The dialyzed Der f and ...

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Abstract

The present invention relates to method for quantification of the absolute amount of allergen in an allergen sample comprising : a) providing a known amount of one or more allergen calibration standard peptide(s) having a sequence of amino acids which is identical with, and optionally unique for, a sequence to be found in the allergen to be quantified and optionally labelling said allergen calibration standard peptide(s), b) degrading the allergen sample to obtain a mixture of peptides, and optionally labelling said peptides with one or more labelling agent(s), wherein at least the peptides in the degraded allergen sample or the calibration standard peptides are labelled, and if both the peptides in the degraded allergen sample and the allergen calibration standard peptide(s) are labelled, the labelling agent(s) used for labelling the allergen calibration standard peptide(s) are different from the labelling agent(s) used for labelling the peptides of the degraded allergen sample, c) quantifying the absolute amount of allergen by correlating the amount of the allergen calibration standard peptide(s) with the amount of the corresponding peptide(s) of the degraded allergen sample by mass analysis.

Description

technical field [0001] The present invention relates to the field of quantification of allergens. Background of the invention [0002] Allergens are antigenic molecules that induce allergic responses and IgE antibody production in humans. They are useful in the diagnosis and treatment of allergies, ie, allergen immunotherapy, which is in the form of allergen vaccines. Allergenic source materials to which humans are exposed, such as food, pollen or mite fecal particles, naturally exist as complex mixtures of primary and secondary allergens. A major allergen is one to which the majority of patients with an allergy to that source react. However, it appears that any protein is a possible allergen, as more and more minor allergens have been identified as awareness has increased. [0003] Due to the complexity of the sources of allergens, the amino acid sequences of several allergens were first deduced from the nucleotide sequences derived from cDNA. Cloning of genes encoding ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6818G01N2458/15
Inventor U·塞帕拉
Owner ALK ABELLO SA
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