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Quantitative measurement kit for glypican and detection method thereof

A technology of phosphatidylinositol and proteoglycan, which is applied in the quantitative detection kit of phosphatidylinositol and proteoglycan, can solve the problems of limiting sensitivity and specificity, and achieve the expansion of detection range, large binding capacity, The effect of shortening the reaction time

Inactive Publication Date: 2017-03-22
北京久峰润达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, enzyme-linked immunoassay (ELISA) is used to detect GPC3, but this method is a chromogenic solid-phase reaction method, and its methodology limits the sensitivity and specificity of detection

Method used

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  • Quantitative measurement kit for glypican and detection method thereof
  • Quantitative measurement kit for glypican and detection method thereof
  • Quantitative measurement kit for glypican and detection method thereof

Examples

Experimental program
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preparation example Construction

[0038] The preparation method of the quantitative assay kit of Glypican (GPC3) of the present invention comprises the following steps:

[0039] The first step: the preparation process of magnetic separation reagent

[0040] 1. Operation steps for preparing magnetic bead buffer, prepare 1L:

[0041] 1. Weigh TRIS 6.06g, NaCl 9.0g, 1.0g TWEEN-20 in a 1L container;

[0042] 2. Use a pipette to measure 0.5ml of Proclin-300 into the above 1L container, then add 800ml of purified water to the above 1L container, and stir thoroughly;

[0043] 3. Adjust the pH meter to measure the pH value, adjust the pH, and control the pH at 8.0 (plus or minus 0.5);

[0044] 4. Weigh 2g of BSA and pour it into the above 1L container;

[0045] 5. Finally, adjust the volume to 1000ml, and filter with a 0.2um filter; after filtering, label it and store it in a cold room at 2-8°C.

[0046] 2. Preparation process of magnetic separation reagent

[0047] 1. Dissolve 2.0mg of disuccinimidyl suberate in...

Embodiment 1

[0087] The magnetic microspheres containing the anti-GPC3 monoclonal antibody of this embodiment were prepared according to the following steps:

[0088] 1. The configuration of the magnetic bead buffer: the formula is shown in Table 1, taking the preparation of 1L as an example:

[0089] 1. Weigh TRIS 6.06g, NaCl 9.0g, 1.0g TWEEN-20 in a 1L container;

[0090] 2. Use a pipette to measure 0.5ml of Proclin-300 into the above 1L container, then add 800ml of purified water to the above 1L container, and stir thoroughly;

[0091] 3. Adjust the pH meter to measure the pH value, adjust the pH, and control the pH at 8.0+0.05;

[0092] 4. Weigh 2g of BSA and pour it into the above 1L container;

[0093] 5. Finally, adjust the volume to 1000ml, and filter with a 0.2um filter; after filtering, label it and store it in a cold room at 2-8°C.

[0094] The magnetic bead buffer is valid for 12 months;

[0095] Table 1 Raw materials and proportioning of magnetic bead buffer

[0096]

[...

Embodiment 2

[0110] The alkaline phosphatase-labeled anti-GPC3 monoclonal antibody of this embodiment was prepared according to the following steps:

[0111] 1. Preparation of enzyme reactant (alkaline phosphatase-labeled anti-GPC3 monoclonal antibody) diluent: the formula is shown in Table 2, taking the preparation of 1L as an example:

[0112] 1. Put 6.06g of Tris, 9.0g of NaCl, 0.05g of Zncl2, 0.5ml of Proclin-300 and 0.05g of MgCl2 in a flask, then add 800ml of purified water into the flask, and stir well to completely dissolve the reagents;

[0113] 2. Adjust PH, control PH in the range of 7.35-7.45;

[0114] 3. Weigh 2g of BSA and pour it into the above beaker;

[0115] 4. Finally, adjust the volume to 1000ml, filter it with a 0.2um filter, label it and store it in a cold room at 2-8°C. The validity period is 12 months.

[0116] Raw materials and proportioning of table 2 enzyme reactant dilution

[0117]

[0118] 2. Coupling of alkaline phosphatase (ALP) and anti-GPC3 monoclona...

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Abstract

The invention discloses a quantitative measurement kit for glypican and a detection method thereof, relates to a kit for measuring or analyzing a material by means of a biologically peculiar combination method and a measurement method thereof, and particularly relates to an immunodetection kit and a detection method thereof. The invention aims at providing a quantitative measurement kit, which is more effective, more sensitive and has higher specificity, for the glypican. The kit provided by the invention comprises a glypican magnetic-separation reagent, an enzyme reactant, a standard substance and a substrate solution. The invention also discloses a preparation method of the kit. The detection method provided by the invention comprises the following steps of sufficiently combining a to-be-detected sample and the standard substance with the glypican magnetic-separation reagent respectively; afterwards, mixing an obtained first mixture with the enzyme reactant, and incubating an obtained second mixture; separating the glypican magnetic-separation reagent; cleaning the glypican magnetic-separation reagent, and then adding the substrate solution into the cleaned glypican magnetic-separation reagent; measuring absorbency. The detection method provided by the invention is good in accuracy, high in precision, high in sensitivity and wide in detection range, and is simple and time-saving to operate.

Description

technical field [0001] The present invention relates to a kit for measuring or analyzing materials by means of a biologically specific binding method and its measuring method, in particular to an immunoassay kit and its detecting method. Background technique [0002] Glypican-3 (GPC3) is a heparan sulfate glycoprotein (heparan suLfate proteoglycan, HSPG) on the surface of the cell membrane. GPG3 is highly expressed in liver cancer tissues, but not in normal liver tissues. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, and most patients are in the middle and advanced stages when they are discovered. HCC has a high morbidity rate and high mortality rate in my country, which seriously endangers the health of the general public, so it deserves great attention and in-depth research. Glypican-3 (GPC3) is a new carcinoembryonic antigen of hepatocellular carcinoma. The GPC3 gene is located on human chromosome x26.1, 900kb in length, with the 5' end facin...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/577
CPCG01N33/535G01N33/577
Inventor 张念林
Owner 北京久峰润达生物技术有限公司
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