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Anti-human glycated albumin monoclonal antibody and use thereof

A monoclonal antibody and glycosylated albumin technology, applied in the direction of anti-animal/human immunoglobulin, application, biochemical equipment and methods, etc., can solve the problems of unsuitable clinical routine development, long detection time, and poor accuracy of immunochemical methods And other issues

Active Publication Date: 2014-02-05
宁波医杰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The determination of GA by HPLC can accurately detect the overall level of blood glucose control in patients in the short term, but it is not widely used because of its high cost and small sample size, which is not suitable for clinical routine
(2) affinity chromatography, (3) colorimetric method and (4) immunochemical method have not been able to be promoted due to the disadvantages of poor accuracy and long detection time.

Method used

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  • Anti-human glycated albumin monoclonal antibody and use thereof
  • Anti-human glycated albumin monoclonal antibody and use thereof
  • Anti-human glycated albumin monoclonal antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 Preparation of anti-human glycated albumin monoclonal antibody hybridoma cells

[0024] Mix complete Freund's adjuvant and human glycated albumin solution at a ratio of 1:1 to make an emulsion. Immunize female Balb / c mice aged 6-8 weeks by intraperitoneal injection (100 microliters per mouse, containing 10 micrograms of human glycated serum albumin). Two weeks later, mice were boosted with 10 micrograms of glycated serum albumin injected intraperitoneally with incomplete Freund's adjuvant. Two weeks later, blood was taken to determine the titer of antibodies. The mice were boosted by injecting 10 micrograms of glycosylated serum albumin through the tail vein. Four days later, the mice were sacrificed, the spleen cells were isolated, and the isolated spleen cells were fused with mouse myeloma cells SP2 / 0 cells with 50% PEG.

Embodiment 2

[0025] Example 2 Screening of anti-human glycated albumin monoclonal antibody hybridoma cells

[0026] Add 100 microliters of human glycated serum albumin dissolved in PBS buffer solution with a concentration of 2 micrograms per milliliter to the wells of a 96-well microtiter plate, and place it overnight at 4 degrees. The next day, wash the 96-well ELISA plate coated with human glycated serum albumin twice with washing solution (PBS buffer containing 0.05% Tween). Then use blocking solution (1%BSA / washing solution) to block the plate for two hours at room temperature. Pour off the blocking solution, then add 100 microliters of cell culture supernatant to the hybridoma culture solution obtained in Example 1 and add it to a 96-well plate, incubate at room temperature for 2 hours, pour off the supernatant, wash with washing solution (containing 0.05% Tween in PBS buffer) to wash the plate twice, then add 100 microliters of alkaline phosphatase-labeled goat anti-mouse IgG antibo...

Embodiment 3

[0027] The cloning culture of embodiment 3 hybridoma cells

[0028] Monoclonal hybridoma cells can be cultured by limiting dilution. The specific method is as follows. The most positive hybridoma cells obtained in Example 2 were counted and serially diluted to prepare a single cell suspension with a concentration of 1 cell / 200 microliters. Add 200 microliters of single cell suspension to the wells of a 96-well cell culture plate (1 cell / 0.2ml / well). Culture in a cell incubator with 5% carbon dioxide at 37°C. About 10 days or so select the monoclonal well, detect the antibody, if positive, clone again, generally clone 3 times. Select clones with strong antibody positive and good cell growth, expand culture, establish lines, and save them.

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Abstract

The invention discloses an anti-human glycated albumin monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein an amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and an amino acid sequence of the light chain variable region is shown in SEQ ID NO:4. The anti-human glycated albumin monoclonal antibody is an IgG1 subtype. The invention also discloses a gene used for encoding the anti-human glycated albumin monoclonal antibody, wherein a nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:1, and a nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:3. The anti-human glycated albumin monoclonal antibody can be used for measuring human glycated albumin.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-human glycated albumin monoclonal antibody, a monoclonal antibody variable region gene and applications thereof. Background technique [0002] Diabetes is a chronic disease. The World Health Organization reported in September 2011 that 346 million people worldwide suffer from diabetes, and the number of people who died from the consequences of high blood sugar was estimated to be 3.4 million in 2004. The latest epidemiological survey shows that there are more than 92.4 million people with diabetes in my country. The national diabetes prevalence rate is 9.7%. my country has become one of the countries with the fastest-growing diabetes prevalence rate in the world. By 2020, India will become the country with the largest number of diabetics in the world. Diabetes as a chronic disease, blood sugar is too high, can cause many complications. Blood sugar monitoring is a...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13G01N33/68G01N33/577
Inventor 赖增祖叶欣徐健杨奎东
Owner 宁波医杰生物科技有限公司
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