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Recombinant adiponectin antigen, antibody and adiponectin nano-latex enhanced immunoturbidimetric assay kit

A technology of recombinant lipid and adiponectin, applied in the direction of anti-hormone immunoglobulin, anti-animal/human immunoglobulin, hormone peptide, etc. Significance, not explaining the normal level and reference range of adiponectin measured, limiting the clinical promotion and application of adiponectin, etc., to achieve the effect of good prediction effect, high accuracy and simple steps

Active Publication Date: 2021-11-12
英诺生物技术(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current international production of recombinant adiponectin by mammalian systems is low in yield and high in cost (yield is about 2mg / L, patent: US 7,365,170 B2), which limits the promotion and application of this recombinant antigen in the detection of adiponectin
[0009] More importantly, most of the current research on adiponectin, such as the above-mentioned patents, did not conduct detailed clinical verification of the reagents produced, did not describe the normal level and reference range of adiponectin measured, and failed to clarify the adiponectin level. The range of abnormal values ​​and the significance of measured abnormal values, and the predictive function of adiponectin for type 2 diabetes and pre-diabetes have not been clinically verified, which further limits the clinical promotion and application of adiponectin detection

Method used

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  • Recombinant adiponectin antigen, antibody and adiponectin nano-latex enhanced immunoturbidimetric assay kit
  • Recombinant adiponectin antigen, antibody and adiponectin nano-latex enhanced immunoturbidimetric assay kit
  • Recombinant adiponectin antigen, antibody and adiponectin nano-latex enhanced immunoturbidimetric assay kit

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Experimental program
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Embodiment 1

[0044] Example 1 Expression of adiponectin antigen

[0045] 1) Recombinant adiponectin antigen expressed by mammalian cells in the prior art

[0046] Reference reports (US 7,365,170 B2; Xu A, et al.Testosterone selectively reduces the high molecular weight form of adiponectin by inhibiting its secretion from adipocytes.J Biol Chem.2005May 6;280(18):18073-80) Antigen expression method. First, using the upstream primer: 5′-GCCCG CGGAT CCATG CTGTT GCTGG GAGCT GTTC-3′ and the downstream primer: 5′-GGCCG CGAAT TCTCA CTTGT CATCG TCGTC CTTGT AGTCG TTGGT GTCATGGTAG AGAAG-3′ from the human adipocyte DNA library (Clontech Corporation, USA) to amplify the gene encoding the adiponectin protein. After treatment with restriction endonuclease BamHI / EcoRI, insert the human adiponectin gene into the expression vector pRSET A, which is stronger than the vector pcDNA3.1 promoter reported in the literature, and insert and express it at the carboxyl end of the adiponectin gene The DNA sequence ...

Embodiment 2

[0049] Example 2 Preparation of Adiponectin Antibody

[0050]Take the natural adiponectin antigen disclosed in the prior art (CN 103777023 B, purchased from Bio-Rad Company, referred to as antigen A, and the antibody produced by it is referred to as antibody A), the recombinant adiponectin antigen of the prior art (US 7,365,170 B2 , referred to as antigen B, and the antibody produced by it is referred to as antibody B), and each 300 μg of adiponectin antigen expressed by mammalian cells of the present invention was emulsified with an equal amount of Freund's complete adjuvant (purchased from Sigma), and multiple points were placed subcutaneously on the back. Inject New Zealand white rabbits of 2.2-2.5kg in a specific pathogen-free (SPF) animal room, and then emulsify the antigen with Freund's incomplete adjuvant (Sigma) every two weeks, and repeat the same method for boosting Immunized 3 times, immunized 4 times in total. Then, 100ml of blood was drawn from the veins of rabbi...

Embodiment 3

[0051] Example 3 Preparation of latex particles coated with anti-human adiponectin polyclonal antibody

[0052] The three kinds of antibodies obtained in Example 2, namely Antibody A, Antibody B and the antibody of the present invention, were coupled to the surface of latex particles by chemical bonding. The latex particles were purchased from Bangslab. group. And using the antibody prepared from the adiponectin antigen expressed by the mammalian cells of the present invention, it is used to optimize the antibody concentration for preparing latex particles.

[0053] Specific steps are as follows:

[0054] 1) 100 mg of carboxyl-containing latex particles were dissolved in 5 ml of MOPS buffer (50 mM, pH 6.4) to obtain a latex particle solution.

[0055] 2) The adiponectin-specific antibody to be cross-linked was dissolved in 5ml of MOPS buffer (50mM, pH 6.4) to a concentration of 1-5mg / ml to obtain an antibody solution; the optimized antibody had a concentration of 3mg / ml.

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Abstract

The invention discloses a recombinant adiponectin antigen, the preparation of which comprises the following steps: a. culturing a cell line highly expressing recombinant human full-length adiponectin protein in a high-sugar DMEM medium, and the culture condition is 0.25-0.75L / min oxygen flow rate, 3-6% carbon dioxide volume, 5-10% bovine serum volume, 100-200rpm speed, so that the cell line reaches 1-3×10 7 b. Replace the medium with serum-free medium and add 0.5% vitamin C to maintain the cell line at 1-3×10 7 The number of cells continued to express adiponectin for 4 to 10 days. The invention also discloses adiponectin antibody, adiponectin antibody nano latex particles and a kit containing the antigen and antibody nano latex particles. The kit adopts the nano latex-enhanced immune turbidimetric method to detect adiponectin with high accuracy, true and reliable data, and good prediction effect on type 2 diabetes and prediabetes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant adiponectin antigen, antibody and adiponectin nano-latex enhanced immune turbidimetry kit and application thereof. Background technique [0002] With the progress of society, changes in the age structure of the population and changes in living habits, the incidence of diabetes has increased rapidly and has become a global epidemic (American Diabetes Association, Diabetes care36Suppl 1:S67-74(2013)). According to the International Diabetes Federation, there are currently more than 380 million diabetic patients worldwide, and 46% of them are not discovered until serious diabetic complications occur. According to statistics, the global annual expenditure on diabetes health care exceeds 548 billion US dollars (International Diabetes Federation, IDF Diabetes Atlas, 6th edition / 2013 update (2013)). According to recent epidemiological surveys, the prevalence of diabetes in my...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/575C07K16/26G01N33/74
CPCC07K14/5759C07K16/26G01N33/74G01N2800/042
Inventor 徐爱民周鹏程钟铃赖华
Owner 英诺生物技术(深圳)有限公司
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