Immunodetection reagent for soluble ST2 (growth stimulation expressed gene 2) protein as well as preparation method and detection method of immunodetection reagent

A growth stimulation and gene expression technology, applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of low measurement accuracy and complicated operation of ST2 detection process, and achieve high sensitivity, accurate quantification and strong specificity Effect

Inactive Publication Date: 2018-06-01
太原瑞盛生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problems of complex ST2 detection process operation and low measurement accuracy in the prior art. The present invention provides a ST2 homogeneous enzyme immunoassay that can detect the ST2 content in the sample to be tested quickly, with high sensitivity Detection reagent and preparation method thereof

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  • Immunodetection reagent for soluble ST2 (growth stimulation expressed gene 2) protein as well as preparation method and detection method of immunodetection reagent
  • Immunodetection reagent for soluble ST2 (growth stimulation expressed gene 2) protein as well as preparation method and detection method of immunodetection reagent

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1: Preparation of glucose dehydrogenase-antigen conjugate

[0043] 1) Preparation of glucose dehydrogenase (GDH) solution:

[0044] a. Weigh 10 mg of GDH with a specification of 100 KU, dissolve it in 8 mL of 50mM PBS solution at room temperature, pH=7.4;

[0045] 2) Activation of ST2

[0046] Add the following chemicals into the beaker and stir to dissolve: 25 mg ST2, 20 mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 5 mg N-hydroxysulfosuccinimide, dissolve Dissolve in 2 mL of 2-(N-morpholine)ethanesulfonic acid with stirring at room temperature, and react for 30 minutes;

[0047] 3) Coupling of GDH and ST2

[0048] a. Add the above-mentioned activated ST2 solution dropwise to the above-mentioned stirred GDH solution;

[0049] b Stir overnight at 2-8°C;

[0050] 4) Purify the conjugated enzyme-labeled antigen

[0051] The conjugated enzyme-labeled antigen was purified by G-25 gel chromatography column to obtain glucose dehydrogenase-ST2 conjugate, wh...

Embodiment 2

[0052] Example 2: Preparation of ST2 Homogeneous Enzyme Immunoassay Reagent

[0053]ST2 homogeneous enzyme immunoassay reagents, including: enzyme-labeled ST2, indicator reagent for detecting ST2 antibody-enzyme-labeled ST2 complex. The indicator reagent is selected from enzyme reagents, radioisotope reagents, fluorescent reagents and chemiluminescent reagents. Preferably, the indicator reagent is an enzyme reagent, including: an enzyme-labeled conjugate and an enzyme substrate. Wherein, the enzyme-labeled conjugate includes glucose dehydrogenase-antigen conjugate, which can be obtained by chemical synthesis.

[0054] Before using the ST2 homogeneous enzyme immunoassay reagent, in order to avoid the reaction between the enzyme-labeled conjugate in the indicator reagent and the enzyme substrate, the enzyme-labeled conjugate and the enzyme substrate are placed separately, so the ST2 homogeneous enzyme The immunoassay reagents consist of two reagents set separately, as follows:...

Embodiment 3

[0063] Example 3: ST2 Homogeneous Enzyme Immunoassay Reagent Reaction Calibration Curve.

[0064] 1) Preparation of ST2 calibrator: Dissolve commercially available human ST2 recombinant protein in a solution similar to human serum matrix (NaCl0.9%, BSA0.2%, NaN 3 0.1%, Tris-HCl pH 7.4), and made different concentrations of calibrators. Taking the ST2 calibrator of Shenzhen Haodi Huatuo Biotechnology Co., Ltd. as the original standard, the ST2 kit was used to test the calibrator of different concentrations 10 times, and the average value was obtained to obtain the concentration of the ST2 calibrator: 10, 20, 37.5 , 75, 150, 300 ng / mL.

[0065] 2) Biochemical analyzer detection: Take the operation of Hitachi 7170 as an example: the measurement wavelength is 340 nm, take different concentrations of calibrator solutions (15 μL), add ST2 R 1 Reagent (160 μL), mix well, then add ST2 R 2 Reagent (40μL), after mixing, measure the OD at different time points 340 The absorbance val...

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Abstract

The invention discloses an immunodetection reagent for soluble ST2 (growth stimulation expressed gene 2) protein as well as a preparation method and a detection method of the immunodetection reagent.The immunodetection reagent comprises enzyme-labeled soluble ST2 protein and an indication reagent which is used for detecting a soluble ST2 protein antibody and enzyme-labeled soluble ST2 protein compound, wherein the enzyme-labeled soluble ST2 protein is prepared from the soluble ST2 protein and glucose dehydrogenase by coupling. With adoption of the immunodetection reagent for the soluble ST2 protein, content of the soluble ST2 protein in samples of human blood and the like can be determined precisely and rapidly. Compared with the detection reagents in the market, the detection reagent hasthe advantages of being convenient, fast, high in sensitivity and specificity, accurate in quantification and the like, and clinical popularization and application are facilitated.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a soluble growth stimulation expression gene 2 protein immune detection reagent and a preparation and detection method thereof. Background technique [0002] Soluble growth-stimulating expression gene 2 protein (ST2) is one of the members of the IL-1 (interleukin 1) receptor family, and the gene expresses two protein products: one with a transmembrane structure, called the transmodel ST2 ( ST2L), a secreted ST2 (sST2) that can be secreted outside the cell. Studies have shown that sST2 is a decoy receptor for IL-33, which can bind to IL-33, thereby blocking the binding of IL-33 to ST2L, and then weakening the cardiovascular protection of IL-33 / ST2L signaling pathway in myocardium under excessive stretch In the process of injury, a large amount of sST2 is produced to make the myocardium lack sufficient IL-33 protection, thereby accelerating myocardial remodeling and ventricular...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/53G01N33/535
CPCG01N33/6869G01N33/5306G01N33/535
Inventor 严芳芳王亚盟程月萍李瑶杜爱铭徐兵
Owner 太原瑞盛生物科技有限公司
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