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Application of ATP as biomarker for evaluating immunization capability and evaluation method thereof

A technology of biomarkers and immune ability, applied in the field of evaluation of immune ability, can solve the problems of cumbersome operation, easy pollution, long experimental time period, etc., to avoid false positive or false negative, convenient, fast and safe operation, and wide detection range Effect

Active Publication Date: 2016-08-03
GUANGZHOU LDEBIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the method for detecting immunity has the following problems: 1. The defect of lymphocyte proliferation detection technology: 3H-TdR incorporation method It represents the total amount of DNA synthesis of cells in a certain amount of culture fluid, rather than the reagent of specific T cells The number is affected by the external culture conditions and there is also the problem of radionuclide contamination; the MTT method is greatly affected by the experimental conditions, and the culture system needs to be strictly controlled; flow cytometry analysis requires certain equipment conditions and the required experimental time period is relatively long. long
2. Defects in T cell secretion function assay technology: Elisa method and Elispot method are more cumbersome and require longer time for detection, and the detected T cells must be able to secrete target cytokines; PCR / RT-PCR has false positive and false negative results , what is not necessarily obtained is the target fragment, and the operation is strict, otherwise it is easy to contaminate. The most important thing is that the latent target fragment cannot be detected by conventional PCR.
3. Defects of T cell-mediated cytotoxic activity assay technology: chromium is radioactive, which is not conducive to safe operation and waste disposal, and it is not quantitatively analyzed and has many operating steps; LDH is a product of the cell's own metabolism, and there may be a phenomenon of spontaneous release, and Effector cells will also be damaged after acting on target cells, and will also release LDH, thus affecting the accuracy of cytotoxic activity determination
[0007] All relevant products and detection methods currently on the market cannot effectively analyze and evaluate the therapeutic effect of patients, and are not suitable for HIV patients or those with low immunity

Method used

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  • Application of ATP as biomarker for evaluating immunization capability and evaluation method thereof
  • Application of ATP as biomarker for evaluating immunization capability and evaluation method thereof
  • Application of ATP as biomarker for evaluating immunization capability and evaluation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] test group:

[0039] The first part: (1) Separation and acquisition of cells (separation by Ficoll method)

[0040] 1. Take a 15ml conical bottom tube, add 6-9ml PBS or RPMI1640, add 3-6ml blood sample, and mix well.

[0041] 2. Take another new 15ml conical bottom tube, add 3-6ml Ficoll separation solution, and carefully add the diluted blood sample to the upper layer of Ficoll to make the layer clear.

[0042] 3. ≤1000°C, 25°C, centrifuge for 20 to 40 minutes, and adjust the Brake to 0.

[0043] 4. Transfer the upper layer of plasma to a new 1.8ml cryopreservation tube and store at -80°C; aspirate the plasma to about 0.5-1cm above the white layer (monocyte layer).

[0044] 5. Carefully transfer PBMCs to a new 1.5ml centrifuge tube; ≤1000g, 20°C-25°C, centrifuge for 4-10min. Add PBS to a total volume of 1ml to resuspend the cells.

[0045] 6. Replace PBS and repeat this step for 3 washes. Trypan blue staining for 1-5min to count the number of cells and viability. ...

Embodiment 2

[0074] Take 6ml of whole blood from 1 normal person and 7 TB patients, separate PBMC by Ficoll method, lyse the cells with 200ulLysis, detect the ATP concentration (ordinate) of the sample, and obtain immunity.

[0075] method:

[0076] 1. Isolation of PBMCs

[0077] ①Use a heparin anticoagulant tube to collect blood (place it at room temperature for no more than 8 hours), and mix it by inverting up and down 8 times before separation to make the cells evenly distributed.

[0078] ② Take a 15ml centrifuge tube, add 5ml RPMI1640 (without FBS), add an equal volume (RPMI1640:blood ratio: 1:1) of blood sample with a Pasteur pipette, and gently mix up and down 8 times to achieve uniform color.

[0079] ③Take another new 15ml centrifuge tube, add 5ml of Ficoll separation solution with an electric pipette gun (the volume ratio of separation solution to blood sample is 1:2), tilt the centrifuge tube at a certain angle, and carefully add the diluted blood sample with a Pasteur pipette....

Embodiment 3

[0095] Take 6ml of whole blood from 1 normal person and 6 TB (tuberculosis) patients, separate PBMC by Ficoll method, lyse the cells with 200ulLysis, detect the ATP concentration (ordinate) of the sample, and obtain immunity. Method is with embodiment 2.

[0096] Such as Figure 5 As shown: the immune function of TB patients is lower than that of normal people, patients 1 and 5 are immunocompromised, patient 4 is discharged after anti-tuberculosis treatment, and the immune function returns to normal, which belongs to normal immunity.

[0097] It can be seen that it is feasible and accurate to obtain the strength of immune ability by detecting the concentration of ATP.

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Abstract

The invention relates to the field of biology, and specially relates to an application of ATP as a biomarker for evaluating immunization capability and an evaluation method thereof. According to the invention, no isotope-labeled material is used, so that defect that the current technologies are not good for safe operation can be overcome; long-time culture of isolated cells for detection is not required, so that the defect of long detection period in the prior art is overcome; rapid detection can be carried out only by cracking of cells, so that the defect of tedious operation in the prior art is overcome, and influence by experiment condition is little. The invention also provides the evaluation method for immunization capability for an aim of non-disease diagnosis, and has the advantages of convenient, fast and safe operation, good sensitivity, high accuracy and wide detectable scope, and an immunization state can be accurately obtained by the results.

Description

technical field [0001] The invention relates to the field of biology, in particular to the use and evaluation method of ATP as a biomarker for evaluating immune ability. Background technique [0002] The material basis of the body's immune function is the immune system. The immune system is composed of immune tissues and organs, immune cells and immune molecules. The immune system can be divided into innate immune system (ie non-specific immune system) and acquired immune system (ie specific immune system). The acquired immune system can generally be divided into two categories: humoral immunity and cellular immunity. Humoral immunity mainly involves antibodies that can bind to various foreign antigens in the human body. They are a class of macromolecular proteins, also known as immunoglobulins, including IgG, IgA, IgM, IgD, and IgE. Cell-mediated immune response (cell-mediated immunity, CMI) mainly involves two types of cells: B cells and T cells, T cells can also be div...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 楼建荣
Owner GUANGZHOU LDEBIO TECH CO LTD