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Quantitative detection method for gamma-interferon and kit

A quantitative detection method and quantitative detection technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of cumbersome operation, waste of manpower, pollution, etc., and achieve the effect of eliminating washing steps, increasing reliability, and simple operation

Inactive Publication Date: 2015-04-29
HEBEI CHEST HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, while the existing kits realize the quantification of interferon-γ, the operation is relatively cumbersome, involving multi-step washing and adding samples, which wastes manpower and is easy to cause pollution

Method used

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  • Quantitative detection method for gamma-interferon and kit
  • Quantitative detection method for gamma-interferon and kit
  • Quantitative detection method for gamma-interferon and kit

Examples

Experimental program
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Embodiment 1

[0029] The quantitative detection method of gamma-interferon of the present invention comprises the following steps:

[0030] 1) Preparation of captured fluorescent microspheres

[0031] In order to achieve the targeting of the capture microspheres, it is necessary to chemically couple the fluorescent microspheres to anti-γ-interferon antibodies (carbodiimide method):

[0032] a. Take 1 μL of 200-220nm fluorescent microspheres (excitation wavelength 488nm) suspension (about 1×10 5 cells / μL) were washed once with washing solution (PBS 0.01mol / L, pH7.4);

[0033] b. Add 80 μL coupling buffer (NaH 2 PO 4 , 0.1mol / l, pH6.2) and 10 μL carbodiimide and NHS (5mg / mL) activators, mix well;

[0034] c. Shake in the dark for 20 minutes at room temperature;

[0035] d. Add 100 μL (4 μg / mL) mouse-derived anti-human γ-interferon antibody (purchased from invitrogen) to the activated microspheres, and mix well;

[0036] e. Shake in the dark for 2 hours at room temperature;

[0037] f. ...

Embodiment 2

[0055] The γ-interferon quantitative detection method in the embodiment of the present invention one, its test result analysis process is as follows:

[0056] 1. Drawing of standard curve

[0057] 1.1 Use the sample diluent to perform a series of doubling dilutions on the interferon-γ standard, the final concentrations are 2000pg / ml, 1000pg / ml, 500pg / ml, 250pg / ml, 125pg / ml, 62.5pg / ml, 31.25 pg / ml, 15.625pg / ml.

[0058] 1.2 Add 50 μl of the above-mentioned standards of different concentrations to sample tubes containing 50 μl of capture microspheres and incubate at room temperature for 1 hour, then add 100 μl of microspheres of fluorescein-labeled anti-human interferon-γ antibody to the above sample tubes , react at room temperature for 1 hour.

[0059] 1.3 Use the CANTOII flow cytometer of BD Company to detect the fluorescence type, count 2000 microspheres, take the FSC / SSC and PE / APC-CY7 scatter diagrams, draw the P1 microsphere gate according to the FSC / SSC scatter diagram...

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Abstract

The invention relates to the technical field of in-vitro diagnosis, and in particular relates to a quantitative detection method for gamma-interferon and a kit. The quantitative detection method for the gamma-interferon disclosed by the invention comprises the following steps: (1) preparing a fluorescent trapping microsphere by adopting a carbodiimide method; (2) releasing the gamma-interferon in vitro; (3) combining the fluorescent trapping microsphere with the gamma-interferon; (4) preparing an antigen-antibody complex; and (5) quantitatively detecting. The quantitative detection kit for gamma-interferon disclosed by the invention comprises the fluorescent microsphere, a coupling buffer solution, carbodiimide, an NHS activating agent and a fluorescein labelled anti-human gamma-interferon antibody. The quantitative detection method for the gamma-interferon and the kit, disclosed by the invention, are simpler to operate; and simultaneously, quantitative detection of the gamma-interferon is realized effectively.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnosis, in particular to a method for quantitatively detecting gamma-interferon and a kit. Background technique [0002] Tuberculosis is one of the serious public health problems worldwide. According to WHO estimates, about 2 billion people in the world are infected with Mycobacterium tuberculosis, and about 2 million people die of tuberculosis every year. my country is one of the 22 countries with a high burden of tuberculosis in the world, and the death toll caused by tuberculosis infection is more than twice that of other infectious diseases. The clinical manifestations of tuberculosis are diverse and can affect various systems of the body, especially extrapulmonary tuberculosis lesions are often very hidden, which brings great difficulties to clinical diagnosis. Traditional TB diagnosis methods are costly and time-consuming, which impose a heavy burden on TB patients and their families. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/5695
Inventor 杨永辉
Owner HEBEI CHEST HOSPITAL
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