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Immunological detection method and kit for detecting mycobacterium tuberculosis

A technology for the detection of Mycobacterium tuberculosis and immunology, which is applied in measurement devices, scientific instruments, analytical materials, etc., can solve problems such as poor sensitivity and specificity, and achieve the effects of simple operation, high sensitivity and good stability

Inactive Publication Date: 2011-11-16
张舒林 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Therefore, the technical problem to be solved in the present invention is to provide an immunological detection method and a test kit thereof for Mycobacterium tuberculosis in view of the poor sensitivity and specificity of existing Mycobacterium tuberculosis immunoassay methods. High specificity and broad application prospects

Method used

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  • Immunological detection method and kit for detecting mycobacterium tuberculosis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] 1. Purification of carbohydrate antigen LAM

[0056] A. Cultivate Mycobacterium tuberculosis H37Ra (ATCC 25177) at 37°C in Roche tuberculosis medium for 3 to 4 weeks, collect the surface culture, and inactivate it in a water bath at 80°C for 1.5 hours;

[0057] B. Wash with PBS, centrifuge to take the precipitate, weigh, suspend with 5ml PBS (pH7.4) per gram of wet bacteria, and ultrasonically break in ice bath: 600W, 6s×6s×90 times.

[0058] C. Centrifuge at 10,000g at 4°C for 30 minutes, and collect the supernatant.

[0059] D. Use a rotary evaporator to evaporate and concentrate at 40°C to 40ml.

[0060] E. Extract three times with an equal volume of phenol / chloroform (1:1, v / v).

[0061] F. The supernatant after phenol / chloroform extraction was extracted twice with 4 times the volume of chloroform / methanol (2:1, v / v), and dialyzed overnight.

[0062] G. Detect sample OD with UV-Vis spectrophotometer 260nm , calculate the nucleic acid content in the sample, diges...

Embodiment 2

[0134] 1. Combined ELISA detection with different coating concentrations 1

[0135] 1) Coating: use 0.05M carbonate coating buffer (NaHCO 3 1.465g, Na 2 CO 3 0.795g, add water to make up to 1000ml, pH9.6) dilute a single antigen to the working concentration, and prepare composition 1, wherein: sugar antigen LAM, 0.5μg / ml; protein 38kDa, 0.8μg / ml; protein CFP10, 0.5 μg / ml; the composition 1 was placed in a 96-well microtiter plate, 50 μl per well, and kept overnight at 4°C.

[0136] 2) Discard the coating solution, wash with PBST 5 times, and spin dry.

[0137] 3) Add 300 μl of PBST containing 1% BSA to each well and incubate at 37° C. for 2 hours.

[0138] 4) Add 100 μl of serum to be tested diluted with PBST containing 1% BSA to each well, and incubate at 37° C. for 0.5 hour.

[0139] 5) Discard the serum, wash with PBST 5 times, and spin dry.

[0140] 6) Add 100 μl horseradish peroxidase-labeled goat anti-human IgG antibody (Jackson Company) diluted in PBST containin...

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Abstract

The invention discloses an immunological detection method and a kit for detecting mycobacterium tuberculosis. According to the immunological detection method, mycobacterium tuberculosis specific antigens are adopted. The mycobacterium tuberculosis specific antigens comprise one or two components selected from protein 38kDa, protein CFP10, carbohydrate antigen LAM, and carbohydrate antigen TBGL. According to the invention, a multiple antigen composition is adopted as a mixed antigen for detecting tuberculosis antibodies. The method and the kit have advantages of high sensitivity, high specificity, and low false positive rate. During a real operation process, a multiple point combined determination result is improved into a single point determination result. Therefore the operation is more convenient. With the method and the kit, antibodies in tuberculosis patient body fluid samples such as serum and hydrothorax can be detected specifically in half a day. The kit can be produced in large scale with low cost. Therefore, the invention has an important significance in the controlling of tuberculosis.

Description

technical field [0001] The invention belongs to the field of biomedical in vitro diagnostic reagents, in particular to an immunological detection method of mycobacterium tuberculosis and a kit thereof. Background technique [0002] Rapid and accurate diagnosis of Mycobacterium tuberculosis (MTB) infection, especially the diagnosis of smear-negative tuberculosis, is of great significance for the control of tuberculosis. There are many diagnostic methods for MTB infection, and the most commonly used clinical method still relies on traditional sputum smear bacteriological examination, but the detection rate is low; MTB culture can be used as the "gold standard" for the diagnosis of tuberculosis, but the culture time is too long , generally 4-6 weeks, it is difficult to meet the clinical needs; Bactec technology shortens the culture time, but the cost is high, and it is difficult to popularize in a short time; X-ray and CT examinations only provide imaging possibilities for diag...

Claims

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Application Information

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IPC IPC(8): G01N33/569C08B37/00
Inventor 张舒林孙战强王洪海
Owner 张舒林
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