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Kit for detecting phosphorylated epidermal growth factor receptor and preparation method thereof

A technology of epidermal growth factor and kit, applied in the field of biomedicine, can solve the problems of cumbersome operation, low sensitivity, expensive instruments, etc., and achieve the effect of overcoming cumbersome operation and less specimen consumption

Active Publication Date: 2013-07-03
RAYBIOTECH INC GUANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In view of the deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a high-throughput, high-sensitivity, high-specificity and low-cost phosphorylated epidermal growth factor receptor detection kit, which uses protein chip technology to detect Simultaneous detection of seventeen phosphorylated epidermal growth factor receptors in multiple samples overcomes the shortcomings of the existing technology, such as cumbersome operation, single detection index, expensive equipment, and low sensitivity. It is cheap, convenient, sensitive, accurate, and high-throughput , Less specimen consumption, can be popularized and scaled up in ordinary laboratories, etc.

Method used

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  • Kit for detecting phosphorylated epidermal growth factor receptor and preparation method thereof
  • Kit for detecting phosphorylated epidermal growth factor receptor and preparation method thereof
  • Kit for detecting phosphorylated epidermal growth factor receptor and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Preparation of a kit for detecting phosphorylated epidermal growth factor receptor.

[0030] In order to detect whether there is the corresponding phosphorylated epidermal growth factor in the sample, prepare the bottom membrane immobilized with specific antibodies against the following proteins: EGFR (Tyr845), EGFR (Tyr992), EGFR (Tyr1045), EGFR (Tyr1068), EGFR (Tyr1086), EGFR(Tyr1148), EGFR(Tyr1173), EGFR(Ser1046 / 1047), EGFR(Ser1070), ErbB2(Tyr877), ErbB2(Tyr1112), ErbB2(Tyr1221 / 1222), ErbB2(Tyr1248), ErbB2( Thr686), ErbB2 (Ser1113), ErbB3 (Tyr1289), ErbB4 (Tyr1284), the above-mentioned various specific antibodies are respectively fixed on the bottom membrane to form independent recognition sites.

[0031] 1. Antibody preparation:

[0032] Specific antibodies against the proteins listed in Table 1 were used, and the sources, batch numbers, and names of the proteins they targeted were detailed in Table 1:

[0033] Table 1 The name of the phosphorylated epi...

Embodiment 2

[0040] Embodiment 2: The experiment of using the kit of the present invention to detect phosphorylated epidermal growth factor receptor.

[0041] After recombinant human epidermal growth factor (rhEGF) stimulates human epithelial tumor cells (A431), the patent kit is used to detect the changes in the content of seventeen kinds of phosphorylated epidermal growth factor receptors in A431. After A431 cells were cultured at 37C, 5% CO2 for 2 to 3 days, the cell density reached about 80 to 90%. Replace the medium with low concentration of calf serum (0.2% FCS) and cultivate overnight. Stimulate with 100 μmg / ml rhEGF. Filter out the cell culture medium, and wash the cells on the wall of the culture dish with cold 1XPBS. Add cell lysate to make the cell number concentration about 2×10 7 Cell count / ml. Mix the cells thoroughly, transfer to a small centrifuge tube, and shake at 2-8°C for 30 minutes. Centrifuge at 4C for 10 minutes at 14,000 rpm, transfer the supernatant to a clean...

Embodiment 3

[0064] Embodiment 3: animal experiments

[0065] Nude mice were used as the experimental model in this experiment. Nude mice were first randomly divided into 4 groups. There are ten mice per group. The first group: control group (control), without any treatment on the mice; the second group: photodynamic therapy (PDT); the third group: cetuximab or Erbitux; the fourth group : photodynamic therapy and cetuximab drug therapy.

[0066] PDT therapy: first inject hypericin into nude mice. After 6 hours, halogen lamp irradiation (wavelength 560-640 nm) was carried out. Cetuximab drug therapy: nude mice were injected intraperitoneally with cetuximab (10 mg / kg). Inject once a day for 90 days. Mice were sacrificed when tumors grew to 2 cm3 or 90 days. Tumor tissue is harvested and disrupted with cell lysate. Then use the patent kit to test the tumor tissue.

[0067] Experimental results:

[0068] (1) Blank control group. The experimental results showed that the content of th...

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Abstract

The invention discloses a kit for detecting phosphorylated epidermal growth factor receptor and the kit adopts the protein chip technology. The kit comprises a base film (1) fixed with several kinds of specific antibodies and reactant and detection agent (2), wherein specific antibodies and phosphorylated epidermal growth factor receptor can perform antibody-antigen reaction, each kind of specific antibodies are fixed on a plurality of independent recognition sites which are fixed on the base film; and the reactant and detection agent (2) are used to detect whether the substance which can perform antibody-antigen reaction with specific antibodies exists in a sample to be detected through the array-ELISA method. The invention also discloses a preparation method of the kit. By adopting the kit of the invention, 17 phosphorylated epidermal growth factor receptors in a plurality of samples can be detected, lots of defects of the prior art can be overcome and the kit has the advantages that the kit is cheap, convenient, sensitive and accurate, has high flux and less amount of sample and can be popularized in the ordinary laboratory and scaled, etc.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a protein chip kit for detecting phosphorylated epidermal growth factor receptor and a preparation method thereof. technical background [0002] Cancer, also known as malignant neoplasm in medical terms, is a disease caused by abnormalities in the mechanism of controlling cell growth and proliferation. In addition to growing uncontrollably, cancer cells also partially invade surrounding normal tissues (invasion) and even transfer to other parts of the body through the internal circulatory system or lymphatic system, which is called cancer cell invasion in the pathology community. Cancer is a multifactorial disease. Its etiology may be related to heredity, gene mutation, aging, or environmental factors. Due to the high morbidity and mortality, it seriously threatens the health of the public. Cancer has become one of the leading causes of death in developed countries. [00...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/53G01N33/531
Inventor 黄若磐黄若春
Owner RAYBIOTECH INC GUANGZHOU
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