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Purification method of tubercle bacilipin Arab mannan and tubercle bacillus diagnostic reagent

A technology of Mycobacterium tuberculosis and mannan, applied in the medical field, can solve the problems of interfering with test results, reducing the specificity of reagents, etc.

Inactive Publication Date: 2011-02-16
SHANGHAI PULMONARY HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some non-specific antigens such as lipomannan (LM), phosphatidylinositol mannoside (PIM), etc. will seriously interfere with the test results and reduce the specificity of the reagents

Method used

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  • Purification method of tubercle bacilipin Arab mannan and tubercle bacillus diagnostic reagent
  • Purification method of tubercle bacilipin Arab mannan and tubercle bacillus diagnostic reagent
  • Purification method of tubercle bacilipin Arab mannan and tubercle bacillus diagnostic reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Experimental materials: Concanavalin agglutinin-Sepharose 4B (COA-Sepharose 4B), lentil agglutinin-Sepharose 4B (LCA-Sepharose 4B) were purchased from GE (formerly Pharmacia), methyl Glucoside was purchased from Sigma, and other reagents were purchased from Sinopharm (Shanghai) Co., Ltd.

[0050] Lipoarabinomannan (LAM Crude) was extracted and purified from Mycobacterium tuberculosis cell wall reference method, lipoarabinomannan (LAM Crude) was dissolved in 20mM Tris-HCl, pH7.6, 0.15M NaCl, 1mM MgCl 2 , 1mM MnCl 2 Balanced, total sugar content (1mg / ml).

[0051] Affinity purification process:

[0052] 1. Column packing: Take 1mL each of COA-Sepharose 4B and LCA-Sepharose 4B and put them into the spin column respectively, with 20mM Tris-HCl, pH7.6, 0.15M NaCl, 1mM MgCl 2 , 1mM MnCl 2 balance.

[0053] 2. Sample loading: add 0.5ml of each sample, centrifuge after 5min, and the centrifuged liquid is Filtration.

[0054] 3. Washing: with 20mM Tris-HCl, pH7.6, 0.1...

Embodiment 2

[0067] Experimental materials: lentil lentil agglutinin (LCA) was self-purified from lentil lentils, and other reagents were purchased from Sinopharm (Shanghai) Co., Ltd.

[0068] Lipoarabinomannan (LAM Crude) in 20mM Tris-HCl, pH7.6, 0.15M NaCl, 1mM MgCl 2 , 1mM MnCl 2 Balance fluid balance;

[0069] Lentil agglutinin (LCA) gold sol labeling: Colloidal gold was prepared by conventional three sodium citrate method, and the optimal marker concentration was selected for labeling of lentil agglutinin to obtain lentil agglutinin (LCA) gold sol labeling substances (without sucrose);

[0070] Lentil agglutinin (LCA) immobilization: Spot lentil agglutinin (LCA) 1mg / mL on a nitrocellulose membrane (NC) strip with a pore size of 0.6um (5mm×3mm), after drying, add 20mM Tris- HCl, pH7.6, 0.15M NaCl, 1mM MgCl 2 , 1mM MnCl 2 3%BSA, 0.05%TWEEN-20 closed;

[0071] Sandwich method detection:

[0072] On a 96-well microplate, mix 20ul lentil agglutinin (LCA) gold sol marker with 20ul ...

Embodiment 3

[0085] Experimental materials: 184 sera, including 78 confirmed tuberculosis sera and 106 non-tuberculosis patient sera.

[0086] Goat anti-human IgG was purchased from Sigma, trisodium citrate, chloroauric acid, potassium carbonate, sodium chloride, bovine serum albumin (BSA), Tween-20, polyethylene glycol and other reagents were purchased from Sinopharm Shanghai Chemical Reagents Co., Ltd., 0.65um Nitrate Trivialin Membrane NC was purchased from AMERSHAM Company. The gold sol and goat anti-human IgG gold sol marker diafiltration reaction kits are provided by Xiamen Bosheng Biotechnology Co., Ltd. The experimental process is as follows:

[0087] 1. Blocking solution preparation: 0.1% BSA / 0.05% Tween-20 / 1% polyethylene glycol pH7.2 phosphate buffer (PBS),

[0088] 2. Preparation of washing solution: 0.05% Tween-20 / 1% polyethylene glycol pH7.2 phosphate buffer (PBS),

[0089] 3. Preparation of gold sol, preparation method of goat anti-human IgG gold sol marker: Boil 100mL...

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Abstract

The invention belongs to the technical field of medical treatment, and in particular relates to a purification method of tubercle bacilipin Arab mannan (LAM) and a tubercle bacillus diagnostic reagent. In the invention, by utilizing the structural differences that the LAM antigen contains three mannose while other impurities (LM, PIM, and the like) only contain one mannose, and the appetency of lectin (such as flat lentils lectin) to three manna branch structures is far higher than two manna and one manna, the LAM antigen purification is carried out by using the lectin to obtain the LAM antigen with the purity of above 95%. The high-purity antigen obtained by purification can form a reagent for LAM antigen detection and can also form a reagent for enriching urine LAM antigen, purifying LAM antibody and detecting the urine LAM antibody.

Description

technical field [0001] The invention belongs to the field of medical technology, and in particular relates to a preparation method of mycobacterium tuberculosis lipoarabinomannan and a diagnostic reagent for mycobacterium tuberculosis. Background technique [0002] LAM antigen Mycobacterium tuberculosis survives and reproduces in the genus through a unique cell wall structure. Its cell wall of Mycobacterium tuberculosis contains various components ( figure 1 ), such as: porin (Porin), polysaccharides, lipids (Lipids), mycolic acid (mycolic acid), phosphatidylinositol mannosides (phosphatidylinositol mannosides.PIM), lipomannans (lipomannans, LM), lipid arabic Mannan (lipoarabinomannan, LAM). Among them, LAM is the main component of mycobacterial cell wall, up to 15 mg / g total weight of bacteria. LAM broadly affects the immune system of an infected host. According to reports, LAM isolated from Mycobacterium tuberculosis and Bacillus leprae can cause T cell proliferation ...

Claims

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Application Information

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IPC IPC(8): G01N33/543C08B37/00
Inventor 胡忠义丁元生崔振玲金瑞良王洁陆俊梅黄晓辰
Owner SHANGHAI PULMONARY HOSPITAL
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