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Preparation of single chain antibody of human liver cancer marker and application thereof

A single-chain antibody and marker technology, applied in the fields of application, botany equipment and methods, biochemical equipment and methods, etc.

Active Publication Date: 2017-05-17
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serological examination mainly detects tumor markers in serum, and currently mainly relies on the detection of AFP, but the sensitivity of AFP in small liver cancer is only about 40%

Method used

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  • Preparation of single chain antibody of human liver cancer marker and application thereof
  • Preparation of single chain antibody of human liver cancer marker and application thereof
  • Preparation of single chain antibody of human liver cancer marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0169] Example 1: Preparation of HGF antibody animal immunization and immune titer determination (taking HGF as an example, the rest of the antibody steps are the same or similar)

[0170] 1) Three 7-week-old female Balb / c mice were first immunized with 100 μg of HGF fusion protein (optionally such as HGF protein with a tagged protein such as His) per mouse, plus an equal volume of Freund's complete adjuvant, Emulsify and mix to form a water-in-oil chylus through a three-way connection, and inject it subcutaneously at multiple points.

[0171] 2) After an interval of three weeks, the dose of antigen was halved, mixed with Freund's incomplete adjuvant and normal saline, and injected intraperitoneally. After four times of immunization in the same way, venous blood was taken to measure the titer.

[0172] 3) Coating HGF protein, 4ng / ml. overnight at 4°C;

[0173] 4) After washing once with PBS, block with 0.2% BSA at room temperature for 1 hour;

[0174] 5) Serum was diluted ...

Embodiment 2

[0178] Example 2: Cell Fusion and Screening of Fusion Cells

[0179] 1) Two 7-week-old Balb / c mice were killed, and macrophages were taken as feeder cells and spread in six 96-well plates;

[0180] 2) The mice three days after intravenous injection were sacrificed, and spleens were taken, fused with myeloma cells SP2 / 0 and plated into six 96-well plates by infinite dilution. The culture condition is DMEM / F12 culture medium containing 20% ​​FBS and 1×HAT;

[0181] 3) Observing the growth of clones after 10 days, supplementing 1×HAT-containing DMEM / F12 culture medium twice during this period;

[0182] 4) Observing the hybridoma cells under a microscope, the round and bright grape clusters can start to detect the specificity. Use the ELISA method to screen the positive wells that can specifically bind to the antigen for subcloning;

[0183] 5) Subcloning by limiting dilution method: the giant cells were taken as cultured cells and spread in six 96-well plates. Adjust the numb...

Embodiment 3

[0186] Example 3: Preparation and purification of ascites

[0187] 1) Prepare 5 female Balb / c mice about 10 weeks old, intraperitoneally inject 500μl pristine / mouse, and after 7-21 days, each mouse intraperitoneally injects 2×10 6 A hybridoma cell in the logarithmic growth phase was observed closely after 10 days, and the ascites was taken out when the mice were killed as much as possible. 3000rpm, 15min centrifugation to remove impurities and grease.

[0188] 2) Precipitation of immunoglobulin by salting-out method: Dilute the ascites water by one time with PBS, and add saturated ammonium sulfate to make the final concentration 33%. Shake while adding dropwise. Place at 4°C for more than 4 hours. Centrifuge at 12000rpm for 15min and discard the supernatant. The precipitate was dissolved with an appropriate amount of PBS, put into a dialysis bag, and dialyzed overnight at 4°C.

[0189] 3) Take out the dialysate, use the swimming pump to put the liquid on the column, the i...

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Abstract

The invention relates to a single chain antibody of a human liver cancer marker, a method for preparing the antibody, a detection reagent and a kit comprising the antibody, and an application thereof. More specifically, the invention relates to the single chain antibody which is specifically combined to the human liver cancer marker, and the human liver cancer marker is selected from the following components: alpha fetoprotein (AFP), glutamyltransferase isoenzyme II (GGTII or GGT2), alpha-L-fucosidase (AFU), hepatocyte growth factor (HGF) and heparin sulfate proteoglycan 3 (GPC3).

Description

technical field [0001] The present invention relates to single-chain antibodies against human liver cancer markers, methods for preparing them, detection reagents and kits containing them, and applications thereof. Background technique [0002] Primary liver cancer (PLC, referred to as liver cancer) is one of the most common clinical malignant tumors. It has an insidious onset, rapid invasion and growth, easy recurrence after treatment, and extremely poor prognosis. It is called the "king of cancer". About half of the liver cancer patients in the world are concentrated in China. About 110,000 people die of liver cancer in China every year, ranking second in the death rate of malignant tumors. At present, surgical resection and liver transplantation are the most effective treatment methods for liver cancer, but the vast majority of liver cancer patients are in the middle and late stages when they see a doctor, and cannot be operated on, and the surgical resection rate is only...

Claims

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Application Information

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IPC IPC(8): C07K16/40C07K16/18C07K16/22C12N15/13C12N15/70C12N15/85
Inventor 张宁罗艺
Owner TIANJIN MEDICAL UNIV
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