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Time-resolved fluoroimmunoassay (TRFIA) kit for heparanase (HPA), and detection method thereof

A technology of time-resolved fluorescence and heparanase, which is applied in the fields of analytical materials, biological material analysis, and measuring devices, etc., can solve problems such as the activity is easily affected by environmental factors, the sensitivity and precision are reduced, and the molecular size of the labeled enzyme is large. Achieve intuitive and reliable results, convenient clinical use, and high sensitivity

Inactive Publication Date: 2014-04-30
朱宝 +7
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ELISA has a large labeled enzyme molecule, its activity is easily affected by environmental factors, which reduces the sensitivity and precision of detection, and the measurement range is narrow

Method used

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  • Time-resolved fluoroimmunoassay (TRFIA) kit for heparanase (HPA), and detection method thereof
  • Time-resolved fluoroimmunoassay (TRFIA) kit for heparanase (HPA), and detection method thereof
  • Time-resolved fluoroimmunoassay (TRFIA) kit for heparanase (HPA), and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of kit and detection of serum samples

[0030] Eu 3+ - Preparation of avidin:

[0031] avidin Eu 3+ , dissolved in 50mmol / L PBS buffer solution with pH7.0 to obtain 5g / L avidin solution, take 1-2mL, convert the buffer condition through PD-10 column, the eluent is containing 0.155mol / L NaCl 50mmol / L Na at pH8.5 2 CO 3 -NaHCO 3 buffer. The protein peaks were collected and quantified by UV absorption analysis (1.46A 280 -0.74A 260 ), dilute avidin to 2g / L with the above eluent. Take 500-1000μL diluted avidin and add Eu containing 0.2-0.4mg 3+ -N 2 -[p-isocyanate-benzyl]-diethylenetriaminetetraacetic acid (Eu 3+ -DTTA) in a vial with magnetic stirring at 30°C for 20 hours. The reaction solution was chromatographed on a Sepharose CL-6B column (1×40cm) equilibrated with 80mmol / L Tris-HCl buffer solution of pH 7.8, A 280 Monitor the collected protein peaks and dilute the aliquots for use.

[0032] Solid-phase antibody preparation on coated p...

Embodiment 2

[0061] Preparation kit: same as Example 1. Preparation of solid-phase antibody on coated plate: Same as Example 1. The preparation of reagent: with embodiment 1. Reagents provided in the kit: The reagents in each box are enough for 48 measurements. The materials in the box are as follows:

[0062] (1), 1 x 48-well plate (4 strips x 12 wells, which can be split into single wells) coated with HPA monoclonal antibody.

[0063] (2), 6×HPA standard solution, 1.0ml / bottle, the concentration of the standard solution is: 0, 0.1, 1, 10, 100, 1000ng / mL.

[0064] (3) 1×biotin-labeled HPA lyophilized product, dissolved in 0.5mL distilled water.

[0065] (4), 1×Eu 3+ - Avidin freeze-dried product, dissolved in 0.5mL distilled water.

[0066] (5), 1× enhancement solution: 15mL.

[0067] (6), 1× washing solution: 30 mL, dilute with distilled water 1:25 when used.

[0068] (7), 1× buffer solution: 30 mL.

[0069] The reagents that should be prepared by the laboratory are the same as i...

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Abstract

The invention relates to a time-resolved fluoroimmunoassay (TRFIA) kit for heparanase (HPA), and a detection method of the TRFIA kit, belonging to the technical field of time-resolved fluoroimmunoassay. The HPA is detected by adopting biotin-avidin system TRFIA on the basis of labeled immunoreaction. Adding HPA standard or sample into a microwell plate coated with a purified HPA monoclonal antibody, and then adding biotin-labeled HPA antigen; adding Eu<3+>- avidin, and washing to remove the non-connected Eu<3+>- avidin after the labeled immunoreactions; adding reinforcement solution, determining the fluorescence intensity cps by using a time-resolved fluorescence instrument, enabling the florescence intensity to be inversely proportional to the concentration of HPA in the sample, thus confirming the content of the HPA in the detected sample comparing with the standard curve. The kit for detecting the HPA, provided by the invention is simple in structure, convenient to use, low in cost, and high in sensitivity, and can be used for detecting the content of HPA in serum, plasma, hydrothorax, ascetic fluid, tissue culture supernate and tissue lysate.

Description

technical field [0001] A time-resolved fluorescent immunoassay kit for heparanase (HPA) and its detection method, specifically involving the detection of heparanase in serum, plasma, pleural effusion, ascites, tissue culture supernatant and tissue lysate (referred to as The detection of HPA) content belongs to the technical field of time-resolved fluorescence immunoassay (TRFIA). Background technique [0002] Heparanase (HPA) is the only hydrolase found in mammals so far that can degrade the side chain of heparan sulfate (HS) on heparan sulfate proteoglycan (HSPG). HPA activation is associated with tumor growth and angiogenesis , inflammation and autoimmunity. Unlike most proteases that cleave polypeptides located in the extracellular matrix, this enzyme tends to degrade the heparan sulfate side chains of heparan sulfate proteoglycans intracellularly. HPA can play a non-enzymatic role independent of extracellular matrix degradation and regulate the extracellular microenvir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
CPCG01N33/573G01N33/582G01N2333/914
Inventor 朱宝谢国强肖华龙黄飚邵科晶许亚丰张艺蒋孟军
Owner 朱宝
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