A kit for detecting akt2 protein and its preparation method

A kit and protein technology, applied in the field of chemiluminescence immunoassay, can solve the problem that the ultra-trace detection of AKT2 protein cannot be satisfied, and achieve the effect of improving the luminescence signal

Active Publication Date: 2016-01-13
SUZHOU HYBIOME BIOMEDICAL ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] The invention overcomes the technical problem that the current chemiluminescence method cannot meet the ultra-trace detection of AKT2 protein, that is, combines the chemiluminescence technology with the biotin-avidin immunomagnification technology and the water-soluble 5,10,15-tris(4-pyridyl )-20-R-carboxyporphyrin is used as a marker to construct a chemiluminescence immunoassay to detect AKT2 protein with high sensitivity, and a kit for detecting AKT2 protein and its preparation method are provided

Method used

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  • A kit for detecting akt2 protein and its preparation method
  • A kit for detecting akt2 protein and its preparation method
  • A kit for detecting akt2 protein and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of the kit for detecting the AKT2 protein of the present invention

[0057] 1. Preparation of calibrator matrix solution

[0058]

[0059] Weigh the above reagents into a clean container, add double distilled water to make up the volume, dissolve and mix well, and measure the pH value. Store at 2-8°C after labeling.

[0060] 2. Preparation of AKT2 protein calibrator

[0061] Prepare calibrator with calibrator matrix solution and pure AKT2 protein, the concentrations are 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100ng / mL, (wherein 0ng / mL is calibrator matrix solution, without AKT2 protein), prepare a series of concentration calibrator, each concentration calibrator is divided into 1mL / bottle with 3mL size vials, freeze-dried, and stored at -20°C.

[0062] 3. Preparation of formula diluent

[0063]

[0064] 4. Preparation of streptavidin-coated solid-phase magnetic particles

[0065] (1) Weigh 250 mg of amino-terminal magnetic particles and p...

Embodiment 2

[0095] Example 2 Prepare the kit for detecting AKT2 protein of the present invention

[0096] 4. Preparation of streptavidin-coated solid-phase plastic particles

[0097] (1) Weigh 250 mg of amino-terminal plastic particles and place them in a beaker;

[0098] (2) Add 5 mL each of methanol and acetone to wash repeatedly 5 times;

[0099] (3) centrifugal separation, discard the supernatant;

[0100] (4) Add 10 mL of 0.2% 1,4-phenylene diisothiocyanate (PDITC) solution dissolved in DMF, place in a constant temperature oscillator, and react for 10 h;

[0101] (5) Wash the reacted plastic particles 6 times repeatedly with acetone and ultrapure water;

[0102] (6) Take the newly prepared plastic particles activated by PDITC and place them in a 30mL centrifuge tube;

[0103] (7) Add 25 mL of PBS, add 4 mg of streptavidin, and place in a constant temperature shaker to react for 20 min;

[0104] (8) Centrifuge, measure the OD value of the solution at 280nm before and after the re...

Embodiment 3～6

[0106] Embodiment 3~6 prepares the kit for detecting AKT2 protein of the present invention

[0107] Monoclonal antibodies were replaced with polyclonal antibodies, genetically engineered antibodies, proteins, macromolecular organic compounds, and the rest were prepared in the same way as in Example 1 to prepare the kit for detecting AKT2 protein of the present invention.

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Abstract

The invention discloses a kit used for detecting AKT2 protein, and a preparation method thereof, and belongs to the field of chemiluminescence immune assay technology. The kit is capable of solving technical problems of existing AKT2 protein ultramicro detection technology. The kit comprises: 1) an AKT2 protein calibration material; 2) a streptavidin-coupled solid phase carrier; 3) a biotinylated AKT2 protein ligand; 4) a 5, 10, 15- tris(4-pyridyl)-20-R-carboxylporphyrin coupling labeled AKT2 protein ligand; and 5) a chemiluminescence substrate of 5, 10, 15-tris(4-pyridyl)-20-R-carboxylporphyrin. The invention also discloses the preparation method of the kit. The kit is mainly used for AKT2 protein ultramicro detection.

Description

technical field [0001] The invention relates to the technical field of chemiluminescence immunoassay, in particular to a kit for detecting AKT2 protein and a preparation method thereof, which combines biotin-avidin immunomagnification technology and chemiluminescence immunoassay technology. Background technique [0002] The AKT2 gene consists of 417 adenines, 526 cytosines, 520 guanines and 386 thymines. AKT2 protein is a protein expressed by the AKT2 gene. AKT2 protein is also known as β-RAC-serine / threonine protein kinase. In the process of scientific research and production, it is necessary to measure the change of AKT2 protein content in the solution to monitor the experimental conditions and monitor the production process. . [0003] Currently AKT2 protein assay methods include colloidal gold immunochromatography (GICA); Western blot (WB); enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA); the advantage of GICA, ELISA, and WB is relatively...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/531G01N21/76
CPCG01N33/532G01N33/54326G01N33/544G01N33/6803
Inventor 常立峻李勇徐海伟胡庆锋陈秀发
Owner SUZHOU HYBIOME BIOMEDICAL ENG CO LTD
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