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Monophosphoinositide proteoglycans-3 chemiluminescence immune analysis determination reagent kit and preparing method thereof

A chemiluminescence immunoassay and phosphatidylinositol technology, which is applied in the field of immunoassay medicine, achieves the effect of wide linear range, rapid detection in large quantities, and easy operation

Inactive Publication Date: 2009-03-04
CHEMCLIN DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, later follow-up reports showed that its C-terminal protein also entered the blood circulation, so the accuracy of the previous patent using the N-terminal protein antibody for GPC3 serological detection was challenged

Method used

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  • Monophosphoinositide proteoglycans-3 chemiluminescence immune analysis determination reagent kit and preparing method thereof
  • Monophosphoinositide proteoglycans-3 chemiluminescence immune analysis determination reagent kit and preparing method thereof
  • Monophosphoinositide proteoglycans-3 chemiluminescence immune analysis determination reagent kit and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of Glypican-3 Chemiluminescence Immunoassay Assay Kit of the present invention

[0036] 1. Configuration of standard products

[0037] Dilute the pure GPC-3 with horse serum to form a calibrator, and the aliquot concentrations are 0ng / ml, 15ng / ml, 50ng / ml, 150ng / ml, 500ng / ml, 1500ng / ml, 6 bottles in total.

[0038] 2. Preparation of antibody-coated plates

[0039] 1. Streptavidin-modified microwell plate: Coat microwells (100 μg / well) with streptavidin at a concentration of 10 mg / L (pH 9.6 0.05 mol / L carbonic acid buffer), overnight at 4°C, the next day Take out, wash 3 times (washing solution uses 0.05% tween20, pH7.4, 0.01mol / L PBS), block with 10% calf serum at 37°C for 30min, and wash 3 times.

[0040] 2. Biotin-labeled GPC3 polyclonal antibody: use biotin succinimide ester to couple with monoclonal antibody under slightly alkaline conditions, fully dialyze with PBS, dilute biotinylated antibody with calf serum, add proclin300, Store below -...

Embodiment 2

[0086] Example 2 The method of using the glypican-3 chemiluminescence immunoassay assay kit of the present invention

[0087] 1. Sample pretreatment

[0088] Take human fasting serum, centrifuge at 2000-4000rpm for 10min, and take the supernatant for analysis without any other special treatment.

[0089] 2. Detection steps

[0090] Before using this kit for detection, it is necessary to take out the packaged medicines in the kit first: the coated plate, enzyme markers, and standard products are allowed to stand at room temperature, and then used after equilibrating to room temperature; after that, place the incubator or water bath Adjust to 37°C; then, prepare a suitable micro-sampler and corresponding tip and check whether the chemiluminescence instrument is working normally.

[0091] The specific operation steps of using this kit to carry out the experiment according to the method of embodiment 1 are as follows:

[0092] 1) Take out the kit and equilibrate to room tempera...

Embodiment 3

[0103] Embodiment 3 Methodological identification of the kit of the present invention

[0104] The test kit prepared in Example 1 is tested according to the conventional manufacturing and testing procedures in the art, and the results are as follows:

[0105] 1. Kit precision

[0106] (1) Standard precision experiment

[0107] Three batches of the kits prepared in Example 1 were taken for the precision test, and 10 kits were taken from each batch. Measure the 150ng / mL GPC3 standard substance 5 times with the kit extracted in Example 1. Calculate the coefficient of variation for the measured concentrations. The measurement results of the three batches of kits in Example 1 are shown in Table 1, and the results show that the coefficient of variation is between 2.0% and 5.5%.

[0108] Table 1 GPC3 standard repeatability experiment

[0109]

[0110] (2) Sample precision experiment

[0111]Precision refers to the reproducibility of the analysis results. Prepare three sample...

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Abstract

The invention relates to the medical field of immunoassay, more specially, the invention provides a chemiluminescent immunoassay detection kit for phosphatidylinositol proteoglycan-3(GPC-3) and a preparation method thereof, and realizes the simultaneous serological detection of GPC-3 N terminal and C-terminal protein with the chemiluminescent immunoassay method. The kit has the advantages of simple sampling, convenient detection and accurate and specific technical method. The invention adopts a biotin-strapavidin system to coat antibodies and improve the efficiency of antibody coating and the linear range of detection as well as sensitivity, and can be conveniently used for the tracing observation of early diagnosis or treatment effect for primary carcinoma of liver.

Description

technical field [0001] The present invention relates to the field of immunoanalysis medicine. Specifically, the present invention provides a chemiluminescent immunoassay kit for fast, accurate and wide range determination of Glypican-3 (GPC3) by a one-step immunoassay method and its Preparation. This kit can accurately and truly measure the protein content of GPC3 in serum samples, including N-terminal protein and C-terminal protein, and can truly reflect the occurrence and development of tumors in patients with primary liver cancer. Background technique [0002] Liver cancer is divided into three types: primary liver cancer (Hepatocellular carcinoma HCC), secondary liver cancer (Cholangiocarcinoma, Ccc) and mixed liver cancer, among which HCC accounts for more than 90%. HCC is the fifth most common cancer in the world, and its fatality rate is the third; liver cancer is mainly distributed in Africa and East Asia. In China, the incidence of liver cancer is three to four ti...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/543G01N21/76
Inventor 张倩云应希堂宋胜利胡国茂郑金来张坤
Owner CHEMCLIN DIAGNOSTICS CO LTD
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