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Method for quantitatively testing MG7-Ag in serum

A quantitative detection method and serum technology, applied in the biological field, to achieve the effect of improving sensitivity, ensuring universality and repeatability, and eliminating inter-chamber differences

Inactive Publication Date: 2009-09-16
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, real-time fluorescent quantitative PCR technology is limited to nucleic acid quantification

Method used

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  • Method for quantitatively testing MG7-Ag in serum
  • Method for quantitatively testing MG7-Ag in serum
  • Method for quantitatively testing MG7-Ag in serum

Examples

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preparation example Construction

[0051] 1) Preparation of tissue homogenate: Wash fresh gastric cancer tissue with normal saline in a water bath or ice bath at 4°C to remove blood stains and dirt, remove capsule or connective tissue, and cut the washed tissue into 0.3-0.5 cm 3 Small pieces, add an appropriate amount of saline, put into the barrel of a mashing machine to make a tissue homogenate. After the tissue homogenate was centrifuged at 3000r / min for 10min, the cells and tissue debris were removed, and the supernatant was kept for use.

[0052] Or cell crushing: collect gastric cancer cell line SGC7901 or MKN45, count the cells, place the cells in liquid nitrogen for 10 minutes, then take them out and thaw them at 37°C, repeat the freeze-thaw operation three times; remove cell debris, and centrifuge at 3000r / min for 10min , and the supernatant was kept for later use.

[0053] 2) Crude extraction of MG7-Ag: Salt out the tissue homogenate or cell lysate supernatant with saturated ammonium sulfate to make ...

Embodiment

[0056] Using MG7-Ag in gastric cancer cell SGC7901 as a standard, the quantitative detection of the MG7-Ag content in the serum to be tested specifically includes the following steps:

[0057] 1) Coating: Divide the wells of the ELISA plate into standard wells and detection sample wells; standard wells: gradient concentration diluted MG7-Ag standard, 100 μl per well; detection sample wells: add 90 μl of serum to be tested and 10 μl of 10 Double-dilute coating buffer, mix well; ELISA plate well overnight at 4°C, discard liquid, wash 3 times with PBS-T solution, 5 min each time.

[0058] Specifically: MG7-Ag in gastric cancer cell SGC7901 was used as a standard, and 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 The concentration gradient dilution of 0 cells was added to the standard wells (A3, B3, C3, D3, E3, F3, G3, H3); to prepare the serum to be tested: draw 2ml of blood, let it stand at room temperature for 30 minutes, 3000rpm for 20 minutes, and draw the upper Set aside a...

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Abstract

The invention belongs to the technical field of biology, and relates a method for quantitatively testing MG7-Ag in serum. The method comprises the following steps: 1) coating: a standard sample and a testing sample; 2) closing: 3) performing the antigen-antibody reaction; 4) performing the second antibody reaction; 5) combining with streptacidin; 6) making the biotinylated DNA report molecular combination; 7) performing the real-time fluorescence quantitative PCR reaction to obtain two groups of PCR amplification curves corresponding to the standard sample and the testing sample; 8) according to the fluoroscopic detection threshold, determining a circular number of the testing sample; 9) according to the fluoroscopic detection threshold, determining a circular number of the standard sample, and drawing a standard curve; and 10) according to the standard curve, determining the content of MG7-Ag in the serum by each testing sample.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method for MG7-Ag in serum, in particular to a quantitative detection method for MG7-Ag in serum. Background technique [0002] Malignant tumor is one of the main diseases that seriously affect human health and threaten human life. The research and application direction of malignant tumors has shifted from focusing on treatment to combining prevention and treatment. Early diagnosis of tumor is particularly important, it determines the success or failure of tumor treatment. Gastric cancer is one of the common major malignant tumors in my country, and its morbidity and mortality continue to rise. At present, the most reliable way for early screening of gastric cancer is endoscopy combined with biopsy pathological examination. However, it is very difficult to identify a small number of gastric cancer cells with atypical morphology, poorly differentiated or undifferentiated ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/577C12Q1/68
Inventor 陈峥樊代明吴开春乔泰东李泉江贴君杜锐
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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