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Method of detecting allergen protein

A protein and allergen technology, applied in the field of protein detection, can solve the problem that there is no known method for detecting allergen protein with high sensitivity, etc.

Inactive Publication Date: 2005-10-26
INC ADMINISTRATIVE AGENCY NAT AGRI & BIO ORIENTED RES ORG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no known method capable of detecting allergen proteins with high sensitivity

Method used

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  • Method of detecting allergen protein
  • Method of detecting allergen protein
  • Method of detecting allergen protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1: Detection of proteins with disulfide bonds in rice extracts

[0107] Grind 20 grams of rice seeds using a mortar. The powder was placed in 400 ml of 1M sodium chloride solution and subsequently agitated for 1 hour, whereby the protein was extracted from the seeds in buffer. After the solution was centrifuged at 14,000 g for 5 minutes to precipitate insoluble components, the supernatant was collected. The supernatant was desalted by dialysis and subsequently freeze-dried. 1 / 80 amount of freeze-dried product (equivalent to 0.25 g of rice seeds) was dissolved in buffer for isoelectric focusing (8M urea, 0.5% CHAPS and 0.1% Bio-Lytes). Iodoacetamide was added to the solution as a SH group protecting agent (final concentration 5 mM), and then the solution was incubated at room temperature for 1 hour. Next, dithiothreitol was added as a reducing agent to the solution (final concentration 5 mM), and then the solution was incubated at room temperature for 1 hour...

Embodiment 2

[0115] Example 2: Analysis of allergen proteins contained in rice extracts

[0116] Will figure 2 Spots 1-6 in B where fluorescent signals were detected were excised from the gel and subsequently digested in-gel with trypsin, thereby fragmenting the proteins in the spots. After HPLC separation of the fragmented proteins, the internal amino acid sequence was determined by the Edman method. The internal amino acid sequence of the protein in each spot thus determined is shown in Table 1. "C" in the internal amino acid sequence shown in Table 1 mRRr " denotes a cysteine ​​residue with a SH group labeled with monobromobimane. Next, each internal amino acid sequence was subjected to homology as a query sequence in amino acid sequence databases (GenBank and PIR) using the FASTA program and the BLAST program Search. The search results showed that the internal amino acid sequence of the protein from each spot was identical or highly homologous to a partial amino acid sequence of ...

Embodiment 3

[0120] Example 3: Detection of proteins with disulfide bonds in pollen extracts

[0121] Ragweed (Ambrosia trifida) pollen was ground with a mortar and pestle in 100 mM Tris-HCl buffer (pH 8.0) containing 1 mM ASF and 1 mM EDTA, and the product was then centrifuged at 14,000 g for 30 minutes. After centrifugation, the supernatant was collected, filtered through an Ultrafree-Cl centrifugal filter (Millipore), and then desalted through a Microcon YM-10 centrifugal filter. The desalted residue was dissolved in buffer for isoelectric focusing (8M urea, 0.5% CHAPS and 0.1% Bio-Lytes). Iodoacetamide was added to the solution as a SH group protecting agent (final concentration 5 mM), and the solution was incubated at room temperature for 1 hour. In addition, dithiothreitol was added as a reducing agent (final concentration 5 mM) and mixed with the solution, and then the solution was incubated at room temperature for 1 hour. In addition, monobromobimane was added as a SH-based label...

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Abstract

The present invention relates to a method for detecting a protein having a disulfide bond or an allergen protein, comprising: protecting by chemically modifying a free SH group of a protein in a sample to be tested; cleaving a disulfide bond of the free SH group-protected protein to expose SH groups; and detecting the exposed SH groups.

Description

technical field [0001] The invention relates to a method for detecting protein, in particular to a method for detecting allergen protein. Background technique [0002] Allergen proteins are widely present in our living environment. Allergen proteins show harmful effects causing food allergy, house dust allergy, hay fever, etc., and sometimes induce fatal symptoms such as anaphylaxis. Extensive screening of allergen proteins is an important goal in order to elucidate the onset mechanism of allergy and to develop a protective approach against allergy. [0003] As a routine method for screening allergen proteins, a detection method based on immunoblotting is used, which involves transferring the protein to a membrane such as nitrocellulose or PVDF, allowing the protein to react with IgE antibodies in the sera of allergic patients, and Antibody binding to the protein is then detected (eg, Weiss, W. et al., Electrophoresis, 18, 826-833 (1977)). When utilizing this method, alle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447G01N33/68
CPCG01N33/6815G01N27/44726
Inventor 矢野裕之黑田秧
Owner INC ADMINISTRATIVE AGENCY NAT AGRI & BIO ORIENTED RES ORG
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