Adiponectin monoclonal antibody, antibody pair and preparation method and application thereof

A monoclonal antibody, adiponectin technology, applied in the biological field, can solve the problem that the sensitivity and precision cannot meet the clinical diagnosis, and achieve the effect of high sensitivity, wide application range and high degree of purity

Active Publication Date: 2021-03-30
CUSABIO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, adiponectin has begun to be used in the detection of type II diabetes and coronary heart disease, but the sensitivity and accuracy of the existing adiponectin detection reagents cannot meet the needs of clinical diagnosis

Method used

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  • Adiponectin monoclonal antibody, antibody pair and preparation method and application thereof
  • Adiponectin monoclonal antibody, antibody pair and preparation method and application thereof
  • Adiponectin monoclonal antibody, antibody pair and preparation method and application thereof

Examples

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preparation example Construction

[0054] The preparation method of the above-mentioned adiponectin monoclonal antibody comprises the following steps:

[0055] Step 1), using a yeast expression system to express adiponectin recombinant protein, the sequence of the protein is SEQ ID NO.1;

[0056] Step 2), separating and purifying the adiponectin recombinant protein obtained in step 1);

[0057] Step 3), immunizing the host animal with the adiponectin recombinant protein obtained in step 2);

[0058] Step 4), obtaining the spleen cells of the immunized host animal obtained in step 3), and preparing fusion cells from the spleen cells;

[0059] Step 5), using the fusion cells prepared in step 4) to prepare adiponectin monoclonal antibody.

[0060] The present invention uses a yeast expression system to express adiponectin recombinant protein, which can successfully realize intracellular expression or secretory expression, and its amplified medium is relatively cheap, the requirements for culture conditions are n...

Embodiment 1

[0085] Adiponectin recombinant protein preparation and separation and purification;

[0086] A. Synthesize the gene sequence of sequence table 1 and the corresponding subcloning primer (primer sequence as described in SEQ ID NO.2 and SEQID NO.3) and connect to pPIC9K plasmid, the connection product obtained is transformed into competent E.coli TOP10, After transformation, spread the bacterial solution on LB solid medium containing 50ug / ml karamycin and 1mg / ml ampicillin, place the culture dish in a 37°C incubator for 12 hours, and select a single colony after PCR and double enzyme digestion to verify that it is correct , perform sequencing, and obtain the recombinants with correct sequencing;

[0087] B. Inoculate the above three correct recombinants into 3ml of LB liquid medium, culture at 200rpm, 37°C for 8-16h, absorb 200ul of bacterial solution under sterile conditions, and inoculate the remaining 2.8ml of bacterial solution into 300ml of LB Liquid medium (containing 50ug...

Embodiment 2

[0093] Preparation of adiponectin monoclonal antibody

[0094] 2.1 Adiponectin recombinant protein immunization mice

[0095] Add 50ug of adiponectin recombinant protein per mouse, add the same amount of Freund's complete adjuvant to make a water-in-oil emulsion, and immunize 6-8 week-old Balb / C female mice; immunize once every two weeks, each time Take 50ug of yeast-expressed recombinant protein and mix it with Freund's incomplete adjuvant. The titer was measured after four times of immunization. If the titer reached more than 1:10000, 50ug / mouse was used for intraperitoneal shock immunization. Three days later, the spleen of the mouse was taken out under sterile conditions and made into a single cell suspension.

[0096] 2.2 Cell Fusion

[0097] Prepare splenocyte suspension and wash it with PBS, then mix it with SP2 / 0 cells, mix and centrifuge according to splenocyte:SP2 / 0=10:1, centrifuge at 1000rpm for 5min, drip dry the mixed cells, tap lightly to loosen the cell mass....

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Abstract

The invention relates to an adiponectin monoclonal antibody and an antibody pair, the adiponectin monoclonal antibody has three kinds, and the three kinds of adiponectin monoclonal antibodies are respectively CSB-DA120AmN (1), CSB-DA120AmN (3) and CSB-DA120AmN (4); and the antibody pair is CSB-DA120AmN (3)-CSB-DA120AmN (4). The invention also provides a preparation method of the adiponectin monoclonal antibody and the antibody pair, and application of the adiponectin monoclonal antibody and the antibody pair in latex enhanced turbidimetry and double-antibody sandwich method immunodetection products, or in preparation of reagents, medicaments or kits for detecting adiponectin. Through gene recombination, the adiponectin monoclonal antibody and antibody pair with more excellent performance are obtained, and the adiponectin monoclonal antibody and antibody pair are stable, efficient, small in batch-to-batch difference and suitable for serving as antibody raw materials of diagnostic reagents.

Description

technical field [0001] The invention belongs to the field of biological technology, and specifically relates to adiponectin monoclonal antibody, antibody pair, preparation method and application thereof. Background technique [0002] Adiponectin (ADPN) is an endogenous biologically active polypeptide or protein secreted by adipocytes. Adiponectin is an insulin-sensitizing hormone (An Insulin-sensitizing Hormone), which can improve insulin resistance (Insulin resistance) and atherosclerosis in mice; studies on humans have found that adiponectin levels can predict type II diabetes and the development of coronary heart disease. [0003] Adiponectin is encoded by the APM1 gene, which is located in the 27th region of the long arm of chromosome 3. There are also susceptibility genes for type 2 diabetes and metabolic syndrome in this region. The total length is 17kb, including 2 introns and 3 Exon, its transcription starts from the 5-terminal cytosine, and the polypeptide coding ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/26C12N15/13G01N33/577G01N33/74
CPCC07K16/26G01N33/577G01N33/74C07K2317/56C07K2317/14Y02A50/30
Inventor 徐珍郑雪松陈莹谭华菊
Owner CUSABIO TECH LLC
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