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Double antibody identification based quantitative detection method of MG7-Ag contained in serum

A quantitative detection method and double antibody technology, applied in the biological field, can solve the problems of affecting the coating efficiency of gastric cancer tumor antigen, affecting the specificity and stability of the method, etc.

Inactive Publication Date: 2010-06-09
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

However, the above method still has certain defects. When the antigen-antibody reaction captures MG7-Ag in the serum, a large amount of non-specific substances in the serum will affect the coating efficiency of gastric cancer tumor-associated antigens; on this basis, after multiple signal conversions and finally After PCR amplification, each link will directly affect the specificity and stability of the method

Method used

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  • Double antibody identification based quantitative detection method of MG7-Ag contained in serum
  • Double antibody identification based quantitative detection method of MG7-Ag contained in serum
  • Double antibody identification based quantitative detection method of MG7-Ag contained in serum

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preparation example Construction

[0072] 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.

[0073] 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.

[0074] 2) Crude extraction of MG7-Ag: Salt out the tissue homogenate or cell lysate supernatant with saturated ammonium sulfate to make ...

Embodiment

[0077] Using tumor cell lysate as a standard, the MG7-Ag content in the serum to be tested for quantitative detection includes the following steps:

[0078] 1) Coating: Dilute the concentration of monoclonal antibody MGb2 to 10 μg / ml with coating buffer, then add to realtime-PCR reaction tubes, add 50 μl to each tube, incubate at 37°C for 1 hour, and wash with PBS-T solution;

[0079] 2) Blocking: add 5 mg / ml BSA to each tube as a blocking solution, incubate at 37°C for 1 hour, and wash with PBS-T solution;

[0080] 3) Antigen-antibody reaction: After the sealing is completed, add 50 μl of standard substance to the standard substance reaction tube as a standard control for detecting the serum to be tested. Specifically, the lysate of gastric cancer cell MKN45 is used as the standard substance:

[0081] After the MKN45 cells were counted, the cells were disrupted to prepare cell lysates, and then 5×10 6 , 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1 , 5, 0 gradient concentr...

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Abstract

The invention relates to a detection method of MG7-Ag contained in serum, belonging to the technical field of biology. The detection method comprises the following steps of: accurately quantitating and detecting the content of the trace antigen MG7-Ag contained in the serum by combining a real-time fluorescence quantitative immune PCR detection method on the basis of antigen signal transformation through the identification and the capture of the MG7-Ag by double antibodies; combining a real-time fluorescence quantitative PCR technology with a specific antigen and antibody reaction system to establish a real-time fluorescence quantitative immune PCR technology, wherein the real-time fluorescence quantitative immune PCR technology identifies the MG7-Ag contained in the serum through the specificity of the double antibodies, then respectively combines biotinylated MG7 with biotinylated DNA through streptavidin, converts a content signal of the MG7-Ag contained in the serum into a nucleic acid signal and then enters a real-time fluorescence quantitative PCR expanding way, thereby accurately detecting the trace antigen MG7-Ag contained in the serum.

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 based on double antibody recognition. 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...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N21/64C12Q1/68
Inventor 陈峥樊代明吴开春聂勇战乔泰东李泉江
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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