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Time-resolved fluorescence immunoassay method fro detecting Dkk-1 and kit thereof

A time-resolved fluorescence and immunoassay technology, applied in analytical materials, biological tests, measurement devices, etc., can solve the problems of poor stability, low sensitivity, and difficulty in meeting Dkk-1 detection, and achieve the effect of improving speed and high sensitivity

Inactive Publication Date: 2009-04-01
RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The ELISA method has the disadvantages of poor stability, large batch-to-batch variation, and low sensitivity, making it difficult to meet the requirements of Dkk-1 detection

Method used

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  • Time-resolved fluorescence immunoassay method fro detecting Dkk-1 and kit thereof
  • Time-resolved fluorescence immunoassay method fro detecting Dkk-1 and kit thereof
  • Time-resolved fluorescence immunoassay method fro detecting Dkk-1 and kit thereof

Examples

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Effect test

Embodiment 1

[0030] Kit: The reagents in each box are enough for 96 measurements, and the materials in the box are as follows:

[0031] 1) 1×96 or 48-well plate (8 or 4 strips×12 wells, which can be split into single wells) microwell reaction plate coated with anti-Dkk-1 monoclonal antibody;

[0032] 2) 6×Dkk-1 standard, lyophilized product, dissolved in 1ml of distilled water before use, the concentrations are 0ng / ml, 12.5ng / ml, 25ng / ml, 50ng / ml, 100ng / ml, 500ng / ml.

[0033] 3) The freeze-dried product of 1×europium-labeled anti-Dkk-1 polyclonal antibody is dissolved in 0.5 ml of distilled water.

[0034] 4) 1× enhancement solution, 15ml.

[0035] 5) 1× washing solution, 30ml, dilute with distilled water 1:25 when used.

[0036] 6) 1X reaction buffer, 30ml.

[0037] in:

[0038] Dkk-1 standard preparation

[0039] Take the commercially available recombinant human Dkk-1 antigen with 0.2% BSA, 0.1% NaN 3 , 50mmol / L, pH7.8 Tris-Hcl buffer solution prepared into 0ng / ml, 12.5ng / ml, 25ng / ...

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Abstract

The invention discloses a time-resolved fluoroimmunoassay used for detecting Dkk1 and a kit thereof. The fluoroimmunoassay includes the following steps: 1. solid-phase anti-body preparing; 2) europium ion marker antibody preparing; and 3) the assay method: based on the double anti-body sandwiched immunoreaction. The invention has the advantages of relatively high sensitivity, specificity and stability on detection of Dkk1; the invention also has a supermatic analytical system which can not only enhance the speed of clinical test results, but also significantly reduce the personal errors and improve the reliability of the test results.

Description

technical field [0001] The invention relates to an immunological detection method for secreted protein dickkopf l (Dkk-1), in particular to a time-resolved fluorescent immunoassay (TRFIA) for detecting Dkk-1 and a corresponding kit. Background technique [0002] The Wnt signaling pathway is an evolutionarily conserved signal transduction pathway that plays an important role in controlling embryonic development, and its inappropriate activation is involved in the occurrence of various human tumors. Some recent studies have reported that gene mutations of the intracellular molecules β-catenin, APC and AXIN involved in this signaling pathway reduce the phosphorylation level of β-catenin in cells and weaken the corresponding ubiquitination degradation, resulting in stable β-catenin The combination of nuclear import and TCF / LEF regulates the expression of related genes, thus leading to the abnormal activation of wnt signaling pathway. The secreted protein dickkopf l (Dkk-1) play...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577
Inventor 盛世乐黄钢谢秀兰
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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