An improved antibody chip kit for simultaneous quantitative detection of multiple cytokines

A cytokine and quantitative detection technology, applied in the field of biomedicine, can solve the problems of cumbersome operation, low sample consumption, low sensitivity, etc., and achieve the effects of increasing stability, reducing background interference, and increasing detection sensitivity.

Active Publication Date: 2016-05-25
RAYBIOTECH INC GUANGZHOU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Aiming at the deficiencies in the prior art, the object of the present invention is to provide an improved body chip kit for quantitatively detecting multiple cytokines at the same time. The kit uses fluorescent detection signals to quantitatively detect dozens of cytokines simultaneously, overcoming the The existing technology has defects such as cumbersome operation, single detection index, and low sensitivity. It has the advantages of low cost, convenience, high throughput, high sensitivity, high specificity, less specimen consumption, and can be popularized and scaled in ordinary laboratories.

Method used

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  • An improved antibody chip kit for simultaneous quantitative detection of multiple cytokines
  • An improved antibody chip kit for simultaneous quantitative detection of multiple cytokines
  • An improved antibody chip kit for simultaneous quantitative detection of multiple cytokines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Screening of the best antibody chip carrier.

[0032] Conventional antibody chips mostly use nitrocellulose membranes as carriers. Because the nitrocellulose membranes are multi-layered structures, it is difficult to wash the chips, so the results of the chips fluctuate greatly. At the same time, because the nitrocellulose membrane chip is not easy to operate on a large scale, the use of large-scale clinical samples is not yet common. Different manufacturers use different methods to fix the nitrocellulose membrane on the surface of the glass slide. Among them, Whatman's SS glass slides, which fix 16 cells containing nitrocellulose membrane on one glass slide, and Gentel has processed it. PATH slides were produced by coating the surface of the slide with nitrocellulose material. In addition, in order to coat the antibody on the slide surface, we screened the carriers activated by different methods. Cy3 and Cy5-labeled streptavidin was spotted on the surface ...

Embodiment 2

[0034] Example 2: Preparation of an antibody chip kit for simultaneous quantitative detection of multiple cytokines.

[0035] In order to detect the presence of the corresponding cytokines in the samples, prepare slides immobilized with specific antibodies against the following proteins: granulocyte-macrophage colony stimulating factor (GM-CSF), interferon gamma (IFNgamma), interleukin 2 (IL-2), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interleukin 8 (IL-8), Interleukin 10 (IL-10), Interleukin 13 (IL -13), tumor necrosis factor alpha (TNFalpha).

[0036] 1. Antibody preparation:

[0037] Specific antibodies against the proteins listed in Table 1 are used. The source, concentration, and protein names of the antibodies are detailed in Table 1:

[0038] Table 1 Name of the antigenic protein targeted by the specific antibody, source and concentration information of the antibody

[0039]

[0040]

[0041] 2. Preparation and storage of antibody chips...

Embodiment 3

[0051] Example 3: Experiment for quantitative detection of cytokines using the kit of the present invention.

[0052] 1. Complete drying of slide chips

[0053] Take the slide chip out of the box, and after equilibrating at room temperature for 20-30min, open the packaging bag, peel off the seal, and then place the chip in a vacuum desiccator or dry at room temperature for 1-2 hours.

[0054] 2. Press image 3 Cytokine standard gradients were serially diluted as shown in

[0055] 2.1. Add 500 μl of sample diluent to the vial of cytokine standard mixture to re-dissolve the standard. Before opening the vial, centrifuge quickly, gently beat up and down to dissolve the powder, and mark this vial as Std1.

[0056] 2.2. Label 6 clean centrifuge tubes as Std2, Std3 to Std7, and add 200 μl of sample diluent to each tube.

[0057] 2.3. Add 100 μl of Std1 to Std2 and mix gently, then extract 100 μl from Std2 and add it to Std3, and dilute to Std7 in this way.

[0058] 2.4. Take 100...

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Abstract

The invention discloses an improved antibody chip kit capable of quantitatively detecting multiple cell factors synchronously. The kit includes a standard tissue slide which is coated with activated amino groups and is used as a surface carrier. Various antigen-specificity antibodies are spotted on a surface of the slide to form a microarray through a non-contacting sample spotting instrument. The slide is divided into sixteen small zones, which are not interfered with each other, through a detachable 2*8-hole plastic frame, wherein each small zone contains an antibody microarray. Each captured antibody is repeated by four times in each antibody microarray. The slide and the frame are tightly clamped with each other through plastic clamping plates which are arranged at the two sides to form an antibody chip. Experimental reagents of a multiple sandwich ELISA reaction are completed on the surface of the slide. By means of the antibody chip kit, multiple cell factors can be quantitatively detected synchronously. A sensitivity of the kit is similar to that of single ELISA but a dynamic detection range of the cell factors is wider. Repeated data, which is as four times as that of ELISA, can be obtained from a single-sample experiment. The kit is high in sensitivity, is high in flux, is little in sample usage amount, is low in cost and is easy to popularize.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to an antibody chip kit for quantitatively detecting multiple cytokines simultaneously. technical background [0002] Cytokines are small-molecule polypeptides (low-molecular-weight proteins) synthesized and secreted by immune cells and non-immune cells of the body that regulate various cellular physiological functions. [0003] Cytokines have a very wide range of biological activities, including promoting the proliferation and differentiation of target cells, enhancing anti-infection and cell killing effects, promoting or inhibiting the expression of other cytokines and membrane surface molecules, promoting inflammatory processes, and affecting cell metabolism. Immune-related cytokines mainly include lymphokines produced by lymphocytes, monokine produced by mononuclear macrophages, interleukin (IL), interferon (IFN), colony stimulating factor (CSF) ), tumor necrosis factor (TNF)...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N21/64
CPCG01N21/6402G01N33/68
Inventor 黄若磐毛应清
Owner RAYBIOTECH INC GUANGZHOU
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