Improved antibody chip kit capable of quantitatively detecting multiple cell factors synchronously

A cytokine and antibody chip technology, applied in the field of biomedicine, can solve the problems of less specimen consumption, cumbersome operation, and low sensitivity, and achieve the effects of reducing background interference, increasing stability, and increasing detection sensitivity

Active Publication Date: 2014-10-22
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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  • Improved antibody chip kit capable of quantitatively detecting multiple cell factors synchronously
  • Improved antibody chip kit capable of quantitatively detecting multiple cell factors synchronously
  • Improved antibody chip kit capable of quantitatively detecting multiple cell factors synchronously

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 membrane as a carrier. Since the nitrocellulose membrane has a multi-layer structure, it is difficult to wash the chip, resulting in large fluctuations in the results of the chip. At the same time, because nitrocellulose membrane chips are 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 immobilize nitrocellulose membranes on the surface of glass slides. Among them are Whatman's SS slides, which fix 16 cells containing nitrocellulose membranes on one glass slide. In addition, Gentel uses processed PATH slides were produced by coating the slide surface with nitrocellulose material. In addition, in order to coat the antibody on the surface of the slide, we screened the carriers activated by different methods. Use a fully automatic spotter to spot Cy3 and Cy5...

Embodiment 2

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

[0035] In order to detect whether there are corresponding cytokines in the sample, 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] Using specific antibodies against the proteins listed in Table 1, the sources, concentrations, and names of the proteins they target are detailed in Table 1:

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

[0039]

[0040] 2. Preparation and storage of antibody chips

[0...

Embodiment 3

[0049] Embodiment 3: Experiment of quantitatively detecting cytokines with the kit of the present invention.

[0050] 1. Complete drying of the slide chip

[0051] Take the slide chip out of the box, and after equilibrating at room temperature for 20-30 minutes, open the packaging bag, peel off the sealing strip, and then place the chip in a vacuum desiccator or dry at room temperature for 1-2 hours.

[0052] 2. Press image 3 Perform serial dilutions of cytokine standard gradients as shown in

[0053] 2.1. Add 500 μl of sample diluent to the vial of the cytokine standard mixture, and redissolve the standard. Before opening the vial, give it a quick centrifuge and gently pipet up and down to dissolve the powder. Label this vial as Std1.

[0054] 2.2. Mark 6 clean centrifuge tubes as Std2, Std3 to Std7 respectively, and add 200 μl of sample diluent to each small tube.

[0055] 2.3. Take 100 μl of Std1 and add it to Std2 and mix gently, then take 100 μl from Std2 and add it ...

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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 molecular polypeptides (low molecular weight proteins) that are synthesized and secreted by the body's immune cells and non-immune cells and regulate a variety of cellular physiological functions. [0003] Cytokines have a 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. Cytokines related to immunity mainly include lymphokines produced by lymphocytes, monokines produced by monocytes and macrophages, interleukin (IL), interferon (IFN), colony stimulating factor (colony stimulating fa...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N21/64
CPCG01N21/6402G01N33/68
Inventor 黄若磐毛应清
Owner RAYBIOTECH INC GUANGZHOU
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