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Novel mass spectrum analysis reagent box and method for detecting heavy hepatitis B

A hepatitis B, kit technology, applied in analytical materials, biological tests, measurement devices, etc., can solve problems such as difficult chemical structures, protein sequence identification and analysis, etc.

Inactive Publication Date: 2008-06-18
许洋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many substances in cells often exist in trace amounts. The current methods for analyzing proteins have limitations in the above aspects. It is difficult to identify and analyze chemical structures and protein sequences with these conventional methods.

Method used

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  • Novel mass spectrum analysis reagent box and method for detecting heavy hepatitis B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 A novel mass spectrometry kit and method for detecting severe hepatitis B

[0071] (1) Experimental method

[0072] 1. Materials

[0073] 1. Specimen source: A. Serum from 50 cases of normal control group; B. Serum from 25 cases of severe hepatitis B patients; C. Serum from 10 critically ill patients with severe hepatitis B (within two days before the death of the patient) ; E. Serum of 10 patients with acute hepatitis B.

[0074] 2. Quality control: A. Human standardized quality control serum B. Mass spectrometer laser energy regulation: Before each test, use the above-mentioned standardized quality control serum.

[0075] 2. Method

[0076] 1. Collection of samples: After the whole blood is collected, draw the serum and store it at -80°C; take out the serum sample in the refrigerator at -80°C, and put it on the ice box to melt; centrifuge at 10,000 rpm and 4°C for 2 minutes; take the supernatant liquid.

[0077] 2. Sample preparation: 2.5 μl of serum is...

Embodiment 2

[0098] Example 2 Identification of β2-microglobulin molecules in blood

[0099] Carbodiimide method (Carbodiimide Method) will have carboxylic acid groups labeled magnetic beads matrix and anti-β2-microglobulin antibody amino groups (Gunn DL, et al.Preparation of sensitive and stable erythrocytes by the carbodiimide method for the detection of primary and secondary IgM and IgG antibody. J Immunol Methods. 1972; 1(4): 381-389.).

[0100] The sample is spotted on a site of an anti-β2-microglobulin antibody matrix.

[0101] Wash with binding buffer. Apply the first wash solution to the site before the sample is completely dry. The wash solution was left on the spot for at least 10 seconds. Thoroughly remove the first wash solution and repeat the above steps with the second wash solution. Thoroughly wash the entire array point with 1% trifluoroacetic acid, elute the biomarkers onto a special metal sheet for mass spectrometry (with 3×3mm round holes), dry the metal sheet natura...

Embodiment 3

[0107] Example 3 Severe Hepatitis B Protein Fingerprint - Sorting Identification of Characteristic Peaks

[0108] The 11729±15Da biomarkers were sorted by multiple-stage mass spectrometry (MS / MS), post-source fragmentation (PSD) and protein ladder sequencing. By breaking molecules into pieces, protein ladders can be generated. This gradient is then analyzed by mass spectrometry. Check the molecular weight of the blood β2-microglobulin molecule in the known cDNA database (molecular weight: 13705.9Da, pI: 6.1). A 11729±15 Da biomarker was identified as variant β2-microglobulin. Its chemical structure is (amino acids arranged from N-terminal to C-terminal):

[0109] Chemical structures of 11729 ± 15 Da biomarkers:

[0110] N-terminal

[0111] IQRTPKIQVYSRHPAENGKSNFLNCYVSGF

[0112] 51 HPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYAC

[0113] 101 RVNHVTLSQPKIVKWDRDM

[0114] C-terminal.

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Abstract

The invention relates to a kit and a novel method of detecting and evaluating the patients with the severe Hepatitis B, and belongs to a non-invasive external detection method. The method utilizes the mass spectrometry to identify and detect a novel variant beta2-microglubolin. The variant beta2-microglubolin or the peak value of 11729 minus and plus 15Da can be used to detect and evaluate the severe Hepatitis B. The rising of the variant beta2-microglubolin or the peak value of 11729 minus and plus 15Da promotes the poor prognosis. The variant beta2-microglubolin level in the serum of the severe Hepatitis B is obviously higher than that of the acute Hepatitis B. The variant beta2-microglubolin level in the serum of people died from the severe Hepatitis B is obviously higher that of the survivor. The method provided by the invention can detect the seriously injured patient, the sensitivity is 100 percent and the specificity is 100 percent. By detecting the peak value of 11729 minus and plus 15Da or the variant beta2-microglubolin captured on the adsorption surface medium of immune body via the quantitative spectrum analysis controlled by the serum of the standardized quality control, the method can be applied in the development of the detection method or the kit for the biological symbol combination in the body fluid separated from the human body. The method has the advantages of accuracy, convenience and rapidness.

Description

technical field [0001] The invention relates to a new method for non-invasive detection and evaluation of severe hepatitis B patient kits, which is characterized in that the β2-microglobulin in the sample that has been captured by magnetic beads or antibodies is accurately identified and detected by mass spectrometry Methods. The method provided by the invention can detect patients with severe hepatitis B, the sensitivity is 100%, and the specificity is 100%. Serum variation of β2-microglobulin level is a good indicator to reflect the degree of liver tissue damage, and it is of great significance for understanding the severity of severe hepatitis and judging the prognosis. Background technique [0002] With the implementation and completion of the Human Genome Project, scientists proposed the concept of the post-genome (post-genome) project, and the focus of research shifted to functional genomics, and the main substance of biological function is protein. In 1994, Wilkins ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/576G01N30/88G01N33/577
Inventor 许洋
Owner 许洋
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