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Detection method and detection kit of soluble ST2

A detection method and soluble technology, applied in the fields of in vitro diagnosis and biological detection, can solve the problems of low concentration of soluble ST2, and achieve the effects of solving sensitivity and specificity, improving accuracy, and improving accuracy

Inactive Publication Date: 2017-12-01
江苏龙维生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Considering that the concentration of soluble ST2 in clinical samples is very low (1 ng / mL-200 ng / mL), and there are structurally homologous similar proteins (such as other proteins of the IL-1 receptor family), the detection of soluble ST2 requires Both sensitivity and specificity are taken into account; while existing methodologies often have defects in specificity or sensitivity

Method used

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  • Detection method and detection kit of soluble ST2
  • Detection method and detection kit of soluble ST2
  • Detection method and detection kit of soluble ST2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Synthesis of IL-33 and its analogs

[0030] Specifically binds IL-33 and its analogues unique to soluble ST2, and its amino acid sequence is as follows:

[0031] Gln-Tyr-Xaa-Cys-Leu-Ala-Leu-Asn-Leu-His-Gly-Leu-Arg-Arg-His-Thr-Val-Arg-Leu-Ser-Arg-Lys-Asn-Pro-Ser- Lys-Glu-Cys-Phe.

[0032] Entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize the following three small peptides, among which XAA are: Asp, Ile, Tyr; the details are as follows:

[0033] (1) Small peptide 1, the amino acid sequence is as follows:

[0034] Gln-Tyr-Asp-Cys-Leu-Ala-Leu-Asn-Leu-His-Gly-Leu-Arg-Arg-His-Thr-Val-Arg-Leu-Ser-Arg-Lys-Asn-Pro-Ser- Lys-Glu-Cys-Phe;

[0035] (2) Small peptide 2, the amino acid sequence is as follows:

[0036] Gln-Tyr-Ile-Cys-Leu-Ala-Leu-Asn-Leu-His-Gly-Leu-Arg-Arg-His-Thr-Val-Arg-Leu-Ser-Arg-Lys-Asn-Pro-Ser- Lys-Glu-Cys-Phe;

[0037] (3) Small peptide 3, the amino acid sequence is as follows:

[0038]Leu-Val-Val-Ser-Tyr-Lys-Tyr-Ala-Leu-Ala-Gln...

Embodiment 2

[0040] Example 2 Fluorescence-labeled IL-33 and its analogs 1

[0041] Cleaning: Take fluorescent microspheres (purchased from Bangs Lab in 2014, item number: 11233) into a centrifuge tube, add 0.1M MES (2-morpholinoethanesulfonic acid) (pH 5.0) buffer to mix, and run at 13000rpm, Centrifuge at 4°C for 15 minutes, discard the supernatant, and resuspend with 0.1M MES (pH 5.0) buffer for use. Activation and cleaning: the activator EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide) and fluorescent microspheres according to the mass The ratio is 2:1:2 for activation, the specific operation is as follows:

[0042] Weigh EDC and NHS and add them into 0.1M MES (pH 5.0) buffer to dissolve, quickly take an appropriate amount into the fluorescent microspheres that have been washed in 4.3.3.1, seal them with a parafilm and place them on a 200rpm shaker at room temperature for 30 minutes, take them out Centrifuge at 13000rpm for 30min at 4°C ...

Embodiment 3

[0048] Example 3 Fluorescence-labeled IL-33 and its analogs 2

[0049] Cleaning: Take fluorescent microspheres (purchased from Bangs Lab in 2014, item number: 11233) into a centrifuge tube, add 0.1M MES (2-morpholinoethanesulfonic acid) (pH 5.0) buffer to mix, and run at 13000rpm, Centrifuge at 4°C for 15 minutes, discard the supernatant, and resuspend with 0.1M MES (pH 5.0) buffer for use. Activation and cleaning: the activator EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide) and fluorescent microspheres according to the mass The ratio is 2:1:2 for activation, the specific operation is as follows:

[0050] Weigh EDC and NHS and add them into 0.1M MES (pH 5.0) buffer to dissolve, quickly take an appropriate amount into the fluorescent microspheres that have been washed in 4.3.3.1, seal them with a parafilm and place them on a 200rpm shaker at room temperature for 30 minutes, take them out Centrifuge at 13000rpm for 30min at 4°C ...

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Abstract

The invention relates to a detection method of soluble ST2. The method comprises the steps as follows: a specific antibody of the soluble ST2 and soluble ST2 ligand protein are combined to serve as detection substances, wherein the ST2 ligand protein is IL-33 (interleukin-33) and analogues thereof. Besides, the invention further provides a kit for detection the soluble ST2 with the method. According to the detection method and the kit for soluble ST2, the IL-33 and analogues thereof can be obtained through gene engineering expression or chemical synthesis, the production process is easier to standardize, and the detection accuracy is further improved.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnosis, in particular to the technical field of biological detection, in particular to a detection method and a detection kit for soluble ST2. Background technique [0002] With the change of lifestyle and the increase of population aging, the number of patients with cardiovascular and cerebrovascular diseases in my country is increasing day by day; A group of complex clinical syndromes, whose main clinical manifestations are dyspnea and fatigue, limited activity tolerance, as well as fluid retention, pulmonary congestion and peripheral edema. Heart failure is the severe and terminal stage of various heart diseases, with high morbidity and mortality, and its five-year survival rate is similar to that of malignant tumors. It is one of the most important cardiovascular diseases today. [0003] At present, there are many difficulties in clinical management of heart failure, such as: the detection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6863G01N33/6887G01N33/6893G01N2800/325
Inventor 韦彦余王军峰徐毅烽王雪根
Owner 江苏龙维生物科技有限公司
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