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Magnetic-bead chemiluminescence kit for detection of tuberculosis infection and application thereof

A technology of magnetic bead chemistry and reagent kits, which is applied in the fields of chemiluminescence/bioluminescence, analysis through chemical reaction of materials, and measuring devices, etc., can solve the problems of limited sensitivity and unsatisfactory effect, and reach the technical level and The effect of low laboratory condition requirements, great clinical application value and high sensitivity

Inactive Publication Date: 2017-08-01
SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing reports show that there are serious non-tuberculous mycobacteria infections in the population, and the effect of the existing commercial kits T-spot. The detection sensitivity is about 80%
The main reason is that the detection methods are ELISPOT and ELISA respectively, the sensitivity of the method is limited, and there are still some deficiencies in the specificity and sensitivity of diagnosis, which need to be improved.
At present, there is no report on the detection kit of tuberculosis infection by magnetic bead chemiluminescence method, which should be further studied

Method used

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  • Magnetic-bead chemiluminescence kit for detection of tuberculosis infection and application thereof
  • Magnetic-bead chemiluminescence kit for detection of tuberculosis infection and application thereof
  • Magnetic-bead chemiluminescence kit for detection of tuberculosis infection and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Sensitivity determination of enzyme-linked chemiluminescence method for detection of interferon-γ.

[0057] 1. Biotin-labeled anti-human gamma interferon monoclonal antibody is the primary antibody, use antibody diluent (containing 1% bovine serum albumin, 0.1M phosphate buffer saline pH7.4, 1M NaCl and 0.02% Procline-300) 1 / 2000 times dilution.

[0058] 2. Alkaline phosphatase-labeled anti-human gamma interferon monoclonal antibody is the secondary antibody, antibody diluent (containing 1% bovine serum albumin, 0.1M phosphate buffer saline pH7.4, 1M NaCl and 0.02% Procline-300 ) 1 / 200 times dilution.

[0059] 3. Use standard diluents for gamma interferon calibration products (standard diluents, 10ml / cartridge, containing 1% bovine serum albumin, 0.1M phosphate buffer at pH7.4 and 0.02% Procline-300) for series Diluted, the concentrations are 400, 100, 25, 6.25, 1.56, 0 pg / ml (picogram / ml) in sequence.

[0060] 4. Add 50 μl primary antibody, 50 μl secondar...

Embodiment 2

[0073] Example 2: Sensitivity and specificity of enzyme-linked chemiluminescence method for detection of tuberculosis infection.

[0074] Sample processing: 3 wells of a cell culture plate are required for each assay sample, and the sample volume required for each well is 1ml, that is, blank control wells, test wells, phytohemagglutinin PHA positive control wells, and serum-free culture medium (AIM- V) 0.1ml, add 0.1ml Mycobacterium tuberculosis-specific polypeptide antigen to the test well, add 0.1ml phytohemagglutinin PHA to the PHA control well, mix each well, place it in a 37°C incubator and incubate for 20 hours, then take 0.1ml of each supernatant, to be tested.

[0075] 1. Take the biotin-labeled anti-human interferon-γ monoclonal antibody as the primary antibody, and dilute the antibody diluent 1 / 2000 times.

[0076] 2. Take alkaline phosphatase-labeled anti-human interferon-γ monoclonal antibody as the secondary antibody, and dilute the antibody diluent 1 / 200 times. ...

Embodiment 3

[0095] The T-SPOT kit was used to detect the same samples for a comparative test. 47 samples of tuberculosis patients and 49 samples of healthy people were tested. The results are as follows

[0096] Table 5 T-SPOT kit detection results of normal people

[0097]

[0098]

[0099] Table 6 T-SPOT kit detection results of tuberculosis patients

[0100]

[0101]

[0102] Known according to experimental result, T-SPOT test kit detection sensitivity is 85.1% (40 / 47), specificity is 87.7% (43 / 49), and test kit detection sensitivity of the present invention is 91.5%, therefore has higher detection sensitivity.

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Abstract

The invention discloses a magnetic-bead chemiluminescence kit for detection of tuberculosis infection. The kit comprises a streptavidin magnetic-bead suspension, a gamma interferon calibrator, phytolectin (PHA), mycobacterium tuberculosis specific antigenic polypeptide, a serum-free culture solution (AIM-V), biotin-labeled anti-human gamma interferon monoclonal antibody, alkaline phosphatase labeled anti-human gamma interferon monoclonal antibody, a luminescent substrate solution (APS 5), a calibrator diluent, an antibody diluent and a cleaning solution. According to the kit, an opaque white enzyme label plate is used for the detection. In comparison with traditional enzyme-linked immunoassay, the kit of the invention has higher sensitivity, is simple and fast to operate, is low-cost, is easy to realize automatic operation, and has a good clinic application prospect.

Description

technical field [0001] The invention relates to biotechnology, in particular to biomedical detection technology, in particular to a magnetic bead chemiluminescent kit for detecting tuberculosis infection and its application. Background technique [0002] Tuberculosis is one of the three major infectious diseases in the world. The number of tuberculosis patients in China ranks second in the world. The national tuberculosis infection rate is 44.5%, with an average of 912,000 new cases and 47,000 deaths per year. Tuberculosis has brought a great economic and social burden to people. With the continuous deepening of research, the clinical diagnosis methods of tuberculosis have made great progress. Immunological detection methods are one of the important auxiliary methods for detecting Mycobacterium tuberculosis infection at this stage. Among them, interferon-γ after antigen-specific stimulation The release experiment (IGRA) is the main one. The IGRA method usually uses the RD ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N33/577G01N33/543
CPCG01N21/76G01N33/54326G01N33/577
Inventor 杨柳张跃建
Owner SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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