Sandwich ELISA quantitative detection method of Golgi protein GP73 and detection kit thereof

A quantitative detection method, Golgi protein technology, applied in biological testing, material inspection products, etc., to achieve high sensitivity and specificity, shorten operation time, and improve sensitivity and specificity

Inactive Publication Date: 2011-03-23
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] According to the data retrieval carried out by the inventor, there are no other report

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  • Sandwich ELISA quantitative detection method of Golgi protein GP73 and detection kit thereof
  • Sandwich ELISA quantitative detection method of Golgi protein GP73 and detection kit thereof
  • Sandwich ELISA quantitative detection method of Golgi protein GP73 and detection kit thereof

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Embodiment 1

[0041] The present invention involves multiple optional monoclonal antibodies whose recognition sites can be seen figure 1 , figure 1 It is a schematic diagram of the GP73 protein structure of the present invention, and the monoclonal antibodies 1F2, 1A6, and 5F10 obtained by the inventors through screening recognize the IV region, and 5B12 and 9D2 recognize the V region.

[0042] In the sandwich ELISA quantitative detection method of Golgi protein GP73 of the present invention, three groups of paired monoclonal antibodies can be selected, namely 5B12-5F10; 5B12-1A6; 5F10-1A6. Select one of the pairing examples as follows, wherein, the coated antibody mAb 1 For 5B12, detection antibody mAb 2 It is HRP-labeled 5F10, namely HRP-5F10.

[0043] ①Use pH 9.6, 0.05mol / L carbonate buffer as the coating solution, dilute the coating antibody 5B12, the working concentration is 1ug / mL, add to 96-well microplate, 100μL / well, moisturize, 4 degrees overnight;

[0044] ②Wash the plate 3 ...

Embodiment 2

[0054] A preferred embodiment of the sandwich ELISA quantitative detection kit for Golgi protein GP73 of the present invention includes coating antibody and detection antibody, blocking solution, coating solution, diluent, chromogen, stop solution, washing solution and standard, including The target antibody and the detection antibody are two monoclonal antibodies against different epitopes in the extracellular region of GP73, and the standard product is the prokaryotic expressed and purified GP73 full-length protein. The detection antibody is a horseradish peroxidase (HRP)-labeled antibody, and the chromogenic reagent is 3,3'5,5'-tetramethylbenzidine (TMB) single-component chromogenic solution. The blocking solution is phosphate buffer containing 5% calf serum, 0.1% Tween20 (Tween20) and 0.01% preservative procline300. The coating antibody and the detection antibody are two monoclonal antibodies directed against different epitopes of the extracellular region of GP73, and the ...

Embodiment 3

[0065] Utilize the method described in embodiment 2, the GP73 content (SGP73) of serum sample is measured, and sample comprises healthy person (70 parts); Non-hepatic patient (131 parts); Hepatitis patient (57 parts); Liver cirrhosis patient ( 60); liver cancer patients (28). For specific results, see image 3 The SGP73 content of hepatitis patients was 110.83±60.33ng / mL, the SGP73 content of liver cirrhosis patients was 124.22±51.28ng / mL, the SGP73 content of liver cancer patients was 135.72±59.21ng / mL, and the SGP73 content of all liver disease patients was 121.18±56.9ng / mL ; The content of SGP73 in patients without liver disease was 62.80±34.91ng / mL, and the content of SGP73 in healthy people was 53.45±17.07ng / mL. Figure 4 is based on image 3 The histogram of the SGP73 content of different liver disease patients, non-hepatic disease patients and normal people, and the comparison of different groups. Figure 4 The data showed that the content of SGP73 in patients with h...

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Abstract

The invention provides a sandwich enzyme-linked immuno sorbent assay (ELISA) quantitative detection method of Golgi protein GP73. Two strains of monoclonal antibodies aiming at different epitopes of a GP73 extracellular domain are used as a coated antibody and a detection antibody; and the GP73 content is detected through substrate developing and a standard curve. A sandwich ELISA quantitative detection kit of the GP73 comprises the coated antibody, the detection antibody, sealing solution, washing buffer, a color reagent, stop solution and a standard substance, wherein the coated antibody and the detection antibody are two strains of monoclonal antibodies aiming at different epitopes of the GP73 extracellular domain; and the amino acid sequences of proteins recognized by the monoclonal antibodies are shown as SEQ ID No. 1 and SEQ ID No. 2 respectively. Through the sandwich ELISA quantitative detection method and the sandwich ELISA quantitative detection kit of the GP73, the sensitivity and the specificity for detecting the GP73 are improved, high stability and reproducibility are guaranteed, the operating time is shortened and the cost is reduced.

Description

technical field [0001] The invention relates to an immunological detection method of serum markers for early diagnosis of liver cancer, in particular to a sandwich ELISA quantitative detection method of Golgi protein GP73 and a detection kit thereof. Background technique [0002] The liver is the largest organ in the human body, and liver disease is also the disease with the highest morbidity and mortality. Due to the strong compensatory ability of the liver, some injuries often have no clinical manifestations, resulting in a strong latent liver disease. When clinical manifestations appear, the liver disease is already at an advanced stage and it is difficult to effectively treat it. At present, there are many kinds of liver function tests that can be carried out clinically, but each test can only detect a certain function of a certain aspect of the liver. Up to now, there are still no indicators that can reflect most of the functions of the liver. Therefore, Often lead to ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 彭涛古艳丽
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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