Kit for detecting glypican-3 and detection method

A technology of phosphatidylinositol and proteoglycan, which is applied in the detection kit and detection field of phosphatidylinositol proteoglycan 3, can solve problems such as poor stability, troublesome preparation of biological enzymes, and enzyme denaturation, and achieve high cost, Good effect, simple method effect

Inactive Publication Date: 2016-08-10
FUZHOU HOSPITAL FOR INFECTIOUS DISEASE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these biological enzymes have poor stability and are easily affected by environmental conditions such as pH and temperature, which can cause enzyme denaturation or inactivation.
Moreover, biological enzymes also have the problems of troublesome preparation, high purification and storage costs, etc.

Method used

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  • Kit for detecting glypican-3 and detection method
  • Kit for detecting glypican-3 and detection method
  • Kit for detecting glypican-3 and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the catalytic property of copper ion and its application

[0029] Preparation of antibody-functionalized copper oxide nanoprobes:

[0030] Add 1mg copper oxide (particle size less than 50nm) to 1mL phosphate buffer (10mM, pH7.4) and ultrasonically disperse evenly. Slowly add 50 μL of 150 μg / mL GPC3 antibody (1G12, Santa Cruz Biotechnology, Inc.), and shake for 3 hours. Add BSA to a final concentration of 1%, and block for 1 h to obtain antibody-functionalized copper oxide nanoprobes (CuO NPs-Ab).

[0031] Meanwhile, copper oxide nanoprobes (CuO NPs-BSA) that only blocked BSA were obtained as a reference according to the above method, omitting the step of antibody modification.

[0032] Catalytic properties of copper ions:

[0033] figure 1 It is the verification diagram of copper ion catalytic activity, a is TMB solution, b is TMB plus H 2 o 2 solution, c is the solution of TMB plus copper ions, d is the solution of TMB plus H 2 o 2 solution with c...

Embodiment 2

[0039] Example 2: Detection of Glypican 3 (GPC3)

[0040] (1) Solution preparation

[0041]Protein coating diluent: 6.7mM phosphate buffer (pH7.4);

[0042] Washing solution: 50mM phosphate buffer (pH7.4) + 135mM sodium chloride + 2.7mM potassium chloride + 0.05% (w / w) Tween-20;

[0043] Blocking solution: 1% (w / w) BSA solution;

[0044] Chromogenic solution: 0.2M acetate buffer (pH4.0)+1.33mM TMB+1.33M H 2 o 2 ;

[0045] Stop solution: 2M sulfuric acid solution.

[0046] (2) Detection method

[0047] Coating: Dissolve and dilute 50 μg of the protein sample to be tested with 200 μL coating diluent, add 50 μL to a 96-well plate and place at 37 °C for 2 hours, discard the liquid in the well, and wash 3 times with washing solution;

[0048] Blocking: Add 200 μL of blocking solution to each reaction well and place at 37°C for 2 hours, and wash 3 times with washing solution after blocking;

[0049] Incubation of nanobiological probes: Dilute the prepared antibody-functional...

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Abstract

The invention relates to a kit for detecting glypican-3 (GPC3) based on catalytic property of copper ions and a detection method. The kit mainly comprises an antibody-functionalized copper oxide nanoprobe and a reagent for detection. The antibody-functionalized copper oxide nanoprobe is prepared by copper oxide nanoparticle surface modification of GPC3 antibody and confining by the use of BSA (Bovine Serum Albumin), wherein mass ratio of the copper oxide nanoparticle to the antibody is 1:0.0015-0.03. The reagent for detection is composed of a coating buffer, a washing liquid, a confining liquid, a developing solution and a stop buffer. The invention mainly has the following beneficial effects: detection of protein is realized by the utilization of catalytic property of copper ions; and the detection method is simple and economical, has good effects, and requires no use of high-cost bio-enzyme which is sensitive to the environment.

Description

(1) Technical field [0001] The invention relates to a kit and a detection method for detecting glypican-3 (glypican-3, GPC3) based on the catalytic properties of copper ions. (2) Background technology [0002] Biological enzyme is a kind of efficient biocatalyst. Due to its high substrate specificity and high catalytic activity, biological enzyme has been widely used in medicine, biotechnology, chemical industry, environmental science and other fields. Peroxidase is a class of biological enzymes that can activate hydrogen peroxide (H 2 o 2 ) undergoes many chemical reactions and is applied in fields such as analysis and diagnosis, such as horseradish peroxidase (HRP). However, these biological enzymes have poor stability and are easily affected by environmental conditions such as pH and temperature, which can cause enzyme denaturation or inactivation. Moreover, biological enzymes also have the problems of troublesome preparation, high purification and storage costs. In r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/31
CPCG01N33/68G01N21/31
Inventor 刘景丰刘小龙郑爱仙黄爱民曾永毅
Owner FUZHOU HOSPITAL FOR INFECTIOUS DISEASE
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