MTB (Mycobacterium Tuberculosis) infection diagnosis kit

A technology for Mycobacterium tuberculosis and a diagnostic kit, which is applied in the field of biomedical testing, can solve the problems that specificity and sensitivity need to be improved, detection efficiency needs to be improved, and detection time is long, so as to achieve high sensitivity and specificity and save energy. Effects of high detection time, specificity and sensitivity

Active Publication Date: 2016-09-21
WUHAN DANGKANG XING ZHONG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specificity and sensitivity of the existing antigen stimuli and kits still need to be improved. At the same time, the entire

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of ELISPOT plate pre-coated with capture antibody

[0048] S1: Take the ELISPOT plate covered with PDVF membrane at the bottom, add 18 μL ethanol with a volume fraction of 70% to each well and incubate at room temperature for 1 min; wash each well with 100 μL sterile PBS solution containing 0.05% Tween-20 for 3 times, and obtain the Pretreated ELISPOT plates;

[0049] S2: Use sterile PBS solution to prepare the anti-human IFN-γ monoclonal antibody into a 15 μg / mL coating antibody solution, add 100 μL of the coating antibody solution to each well of the pretreated ELISPOT plate as described in step S1, and incubate at 4 °C 10h;

[0050] S3: Discard the coating antibody solution, wash each well with 200 μL sterile PBS solution containing 0.05% Tween-20 for 5 times, and dry it on sterilized absorbent paper for the last time;

[0051] S4: Add 200 μL of blocking solution to each well and incubate at 4°C for 10 h; discard the blocking solution, wash tw...

Embodiment 2

[0053] Embodiment 2 The use method of the Mycobacterium tuberculosis infection diagnostic kit of the present invention

[0054] The Mycobacterium tuberculosis diagnostic kit of the present invention comprises the following components: an ELISPOT plate pre-coated with the monoclonal antibody of human IFN-γ prepared in Example 1, an insulin-like growth factor, and biotin-labeled anti-human IFN -γ antibody, horseradish peroxidase-labeled streptavidin, 3-amino-9-ethylcarbazole chromogenic solution, antibody diluent and positive control stimulus.

[0055] Wherein, the antigen stimulus is selected from one or more polypeptides in the sequence shown in SEQ ID NO.1-12; the antibody diluent is a PBS solution containing 1% bovine serum albumin; the positive control stimulus One selected from staphylococcal enterotoxin B, phytohemagglutinin and international standard polypeptide pool CEF.

[0056] Additional reagents to be prepared include fetal calf serum, cell culture medium, DMSO and...

Embodiment 3

[0071] Example 3 Mycobacterium tuberculosis infection diagnostic kit of the present invention is applied to the clinical diagnosis of suspected patients with Mycobacterium tuberculosis

[0072] 1. Test purpose: To test the clinical applicability, specificity, sensitivity and coincidence rate with other diagnostic techniques of the Mycobacterium tuberculosis infection diagnostic kit of the present invention in tuberculosis diagnosis. Wherein, in the kit, the antigen stimulus is a combination of SEQ ID NO.5-9 polypeptides of the present invention; the positive control stimulus is phytohemagglutinin.

[0073] 2. Test method: Collect blood samples from 78 highly suspected patients with Mycobacterium tuberculosis infection, each 2-5mL, use Ficoll lymphocyte separation medium to separate peripheral blood mononuclear cells (PBMC), PBMC through 5-10mL cell culture medium After washing twice, resuspend in cell culture medium containing 10% fetal bovine serum, count the cells, and then ...

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Abstract

The invention belongs to the field of biomedicine examination, and relates to an MTB (Mycobacterium Tuberculosis) infection diagnosis kit. The MTB infection diagnosis kit provided by the invention is prepared from the following components: an antigen stimulant, a pre-coated ELISPOT (Enzyme-Linked Immunospot Assay) plate of a capture antibody, an insulin-like growth factor, a detection antibody, HRP (Horse Radish Peroxidase)-labeled streptavidin, a 3-amino-9-ethyl carbazole developing solution, antibody diluent and a positive control stimulant; the antigen stimulant is selected from one or multiple polypeptides in sequences as shown in SEQ ID NO.1 to 12. The MTB infection diagnosis kit provided by the invention is high in sensitivity and specificity and stable in property; meanwhile, when the MTB infection diagnosis kit provided by the invention is applied to in-vitro detection on MTB, the detection time can be saved, the detection steps can be simplified, and the detection efficiency can be increased.

Description

technical field [0001] The invention belongs to the field of biomedical testing, in particular to a diagnostic kit for mycobacterium tuberculosis infection. Background technique [0002] Tuberculosis is one of the most important infectious diseases that threaten human health, and China is one of the 22 countries with severe tuberculosis epidemics in the world. The annual incidence of tuberculosis is nearly 1.3 million, accounting for 14.3% of the global tuberculosis incidence, ranking No. 1 in the world. Two, second only to India. The fifth national tuberculosis epidemiological survey shows that there are currently more than 5 million active tuberculosis patients in my country, and more than 500 million people are infected with Mycobacterium tuberculosis, accounting for 45% of the country's total population. In the latest 10-year epidemiological statistics, it was found that the death rate of tuberculosis is second only to AIDS. [0003] The clinical manifestations of tube...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/569
CPCG01N33/5695G01N33/577G01N33/68G01N2333/35
Inventor 陈立国
Owner WUHAN DANGKANG XING ZHONG BIOTECHNOLOGY CO LTD
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