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Human adiponectin ELISA kit

A kind of adiponectin and kit technology, applied in the field of medical immunology human adiponectin monoclonal antibody engineering, can solve the problems of high price, long operation time, and unfavorable large-scale detection

Inactive Publication Date: 2012-06-27
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The adiponectin ELISA kit is expensive and takes a long time to operate, which is not conducive to large-scale detection of human adiponectin content

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Cloning Construction and Sequence Analysis of Human Adiponectin Gene

[0042] experiment material:

[0043]Horseradish peroxidase (HRP) is a product of Shanghai Sangong, and plasmid pMD 18-T, TaqDNA polymerase, Kpn I enzyme, EcoRI enzyme, T4 DNA ligase and DNAMarker are products of Dalian Bao Biological Company. Plasmid small mention medium extraction kit and DNA gel recovery kit are products of Omiga Company. The vectors pCDNA3.1(+) and Trizol are products of Invitrogen Company. M-MLV reverse transcriptase is a promega product. Mouse anti-human adiponectin monoclonal antibody was purchased from CST Company; Ni-NTA purification column Qiagen Company; human embryonic kidney cells (human embryonic kidney cells, HEK293) were purchased from American Type Culture Collection (ATCC); and DMEM high pond The culture medium and fetal bovine serum are products of Hycolon; the transfection reagent FuGENE HD is a product of Roche; dithiothreitol (DTT), DEPC water, G418,...

Embodiment 2

[0059] Embodiment 2 Preparation of recombinant human adiponectin

[0060] 1. Extraction of plasmid (pcDNA3.1-APN): Take a small amount of plasmid-containing glycerol bacterial solution stored at -80°C, draw on LB plate (100μg / ml ampicillin), activate overnight at 37°C, pick a single clone, and inoculate in 3ml LB medium (100 μg / ml ampicillin) was cultivated overnight at 37°C, and 1 mL of the culture solution was taken to inoculate fresh LB medium (100 μg / ml ampicillin) at a ratio of 1:50, and cultured at 37°C for 12 hours. The bacteria were collected by centrifugation, and the plasmid (pcDNA3.1-APN) was extracted using a plasmid extraction kit. The concentration was determined by UV spectrophotometer.

[0061] 2. Recombinant pcDNA3.1-APN stably transfected human embryonic kidney cells (human embryonic kidney cells, HEK293): HEK293 cells were cultured in a 35mm cell culture dish to 80% confluence (3-6×10 5 cells / 35mm Petri dish). According to the instructions of FuGENE HD Tr...

Embodiment 3

[0070] Embodiment 3 Preparation of mouse anti-human adiponectin monoclonal antibody

[0071] Taking XA-187-1 as an example, the preparation steps of mouse anti-human adiponectin monoclonal antibody polymer are as follows:

[0072] Step 1, animal immunization

[0073] Dissolve 150 μg of purified human recombinant adiponectin protein in 1.5 ml of normal saline, mix it with an equal amount of Freund’s complete adjuvant (purchased from Sigma) and fully emulsify it, inject BALB / c mice subcutaneously in multiple points on the back, and immunize 3 mice Eight-week-old female BALB / c mice were immunized for the second and third times at intervals of 4 weeks and 3 weeks, in which Freund's incomplete adjuvant was used for the second immunization, and no adjuvant was added for the third immunization. The immunization route was intraperitoneal injection. 10 days after the third immunization, the indirect ELISA method was used to detect the serum titer (enzyme-cleaved and purified protein co...

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Abstract

The invention relates to human adiponectin monoclonal antibody engineering in the field of medical immunology and relates to preparation of a human adiponectin ELISA kit by employing a hybridoma cell strain XA187 No.1 and a hybridoma cell strain XA187 No.19, and application of the kit to detection of human adiponectin content.

Description

technical field [0001] The invention relates to medical immunology human adiponectin monoclonal antibody engineering, and relates to the preparation of human adiponectin ELISA kit and the application of the kit in detecting the content of human adiponectin. Background technique [0002] Adiponectin (APN) is synthesized and secreted by adipocytes, an adipocyte factor that interacts with the extracellular matrix, also known as Acrp30, apM1, AdipoQ, GBP28. Human adiponectin monomer consists of 244 amino acids, including 3 regions: amino-terminal signal sequence, collagen domain and carboxy-terminal globular domain of APN (gAd). Adiponectin exists in blood in three subtypes: trimer (65kDa), hexamer (150kDa) and high molecular weight (HMW, 18-36 monomers, >280kDa) polymer. [0003] The concentration of adiponectin in serum ranges from 2 to 30 μg / mL, accounting for 0.01% of the total plasma protein, and there is no circadian rhythm change. Cross-linking reaction and sucrose g...

Claims

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Application Information

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IPC IPC(8): C12N5/20G01N33/577C12N15/11
Inventor 陶凌屈延廉坤杨璐金伯泉
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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