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Method for detecting dust mite allergen proteins Der f1 and Der p1 and nitrification products thereof in dust

A technology of allergen protein and detection method, which is applied in the field of detection of dust mite allergen proteins Der f1 and Der p1 and their nitration products in dust, to achieve the effect of improving accuracy and flux and ensuring accuracy

Active Publication Date: 2020-08-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the detection of dust mite allergen proteins and their nitration products in dust samples by HPLC-MS / MS.

Method used

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  • Method for detecting dust mite allergen proteins Der f1 and Der p1 and nitrification products thereof in dust
  • Method for detecting dust mite allergen proteins Der f1 and Der p1 and nitrification products thereof in dust
  • Method for detecting dust mite allergen proteins Der f1 and Der p1 and nitrification products thereof in dust

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 sample analysis process

[0027] 1.1 Selection of characteristic polypeptides of dust mite allergen protein

[0028] Take 10 μg dust mite allergen standard protein (Der f1 and Der p1) solution in a 1.5mL low-protein adsorption tube, add 20 μL acetonitrile, 20 μL 50 mmol·L -1 NH 4 HCO 3 Buffer solution, placed at 37°C for 15 min; add 5 μL reducing solution (45 mmol L -1 DTT and 20mmol·L -1 TCEP dissolved in 50mmol·L -1 NH 4 HCO 3 buffer), placed at 37°C for 30min; add 5μL of 100mmol·L -1 IAA solution, mix and place for 30min at 37°C in the dark; add 5.5 μL 90mmol L -1 DTT stock solution was used to quench the residual IAA; add 134.5 μL of ultrapure water; add 5 μL of trypsin (20 ng·μL -1 , diluted in HCl) so that the enzyme / substrate ratio was 1:100 (w / w). After incubating at 37°C for 24 hours, add 2 μL of 10% formic acid / water (v / v), vortex and mix well, and place at room temperature for 10 minutes; ) solution to redissolve. Then 14000r·min at 4°C ...

Embodiment 2

[0054] Embodiment 2 method reliability evaluation

[0055] 2.1 Solution preparation

[0056] The peptide mixed standard solution configuration is as follows:

[0057] (1) Use 11% acetonitrile / water (v / v) solution to prepare a concentration of 1mg·mL -1 AR-8 stock solution, dilute the single standard stepwise to 100μg·mL -1 , 10μg·mL -1 , 1 μg·mL -1 ; Prepare 1 mg·mL with 15% acetonitrile / water (v / v) solution -1 The SR-12 mother liquor and NO 2 - SR-12 stock solution, dilute the single standard stepwise to 100 μg mL -1 , 10μg·mL -1 , 1 μg·mL -1 ; Use 25% acetonitrile / water (v / v) solution to prepare a concentration of 1 mg·mL -1 NO 2 -AR-8 stock solution, dilute the single standard stepwise to 100 μg·mL -1 , 10μg·mL -1 , 1 μg·mL -1 ;

[0058] (2) High-concentration peptide mixed standard solution: take 100 μL of 1 μg·mL respectively -1 AR-8, SR-12, NO 2 -AR-8 and NO 2 - SR-12 The four peptides are single-labeled in a low-protein adsorption tube and diluted with ...

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Abstract

The invention discloses a method for detecting dust mite allergen proteins Der f1 and Der p1 and nitrification products thereof in dust. The invention provides the detection method capable of simultaneously determining two main dust mite allergen proteins Der f1 and Der p1 and the nitrification products Nitrated Der f1 and Nitrated Der p1 thereof in the dust. The dust mite allergen proteind and the nitrification products thereof in a dust sample are digested into small-molecular polypeptide through enzymolysis; one polypeptide with the highest abundance is selected as the characteristic polypeptide of the dust mite allergen proteins and the nitrification products of the dust mite allergen proteins, qualitative and quantitative analysis is conducted on the characteristic polypeptide throughultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), and qualitative and quantitative analysis is conducted on the dust mite allergen proteins and the nitrification products of the dust mite allergen proteins through conversion. The method is good in selectivity and high in accuracy and analysis flux, and overcomes the defects that a traditional ELISA (enzyme-linkedimmunosorbent assay) method is low in detection flux, cross reaction of structural analogues possibly exists, and nitroproteins cannot be accurately quantified.

Description

technical field [0001] The invention belongs to the field of detection of allergen proteins for environmental health, and is a method for selecting the main allergen proteins Der f1 and Der p1 of dust mite by using ultra high performance liquid chromatography-mass spectrometry (UPLC / ESI-MS / MS) The nitrification products Nitrated Der f1 and Nitrated Der p1 were used as the research objects, and a quantitative detection method was established that could simultaneously determine the content of two major dust mite allergen proteins and their nitrification products (a total of four allergen proteins) in dust. Background technique [0002] With the improvement of industrialization, allergic diseases such as asthma, allergic rhinitis, and eczema are increasing year by year all over the world. The World Health Organization (WHO) has listed allergic diseases as "21st century key research and prevention disease". At present, a large number of studies around the world have confirmed t...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/36G01N30/46G01N30/72
CPCG01N30/02G01N30/06G01N30/34G01N30/36G01N30/461G01N30/7266G01N2030/045
Inventor 杨方星徐帆
Owner ZHEJIANG UNIV
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