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Detection test strip for chemerin and derivative polypeptide thereof and preparation method of detection test strip

A chemokine and test strip technology, applied in the field of medical testing, can solve the problems of high cost, expensive ELISA board, uncertain waiting time for multiple patients, etc.

Active Publication Date: 2017-12-08
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] 2) time consuming
The Elisa plate is generally a 96-well plate, and the whole plate is expensive. It is more economical to wait for enough samples from multiple patients to be tested together. It is not suitable for the detection of single or several patient samples, and the waiting time for multiple patients is uncertain;
[0020] 3) Inconvenient
To read the signal through a specific instrument, it can only be tested in qualified places, such as hospitals and social health
[0021] 4) Expensive
The cost of the ELISA plate is high, it requires a low temperature storage environment, and it is necessary to use an instrument to read the results

Method used

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  • Detection test strip for chemerin and derivative polypeptide thereof and preparation method of detection test strip
  • Detection test strip for chemerin and derivative polypeptide thereof and preparation method of detection test strip
  • Detection test strip for chemerin and derivative polypeptide thereof and preparation method of detection test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1C-23

[0085] The preparation of embodiment 1C-23 antibody and recombinant Chemerin protein antibody:

[0086] (1) Use the ReadiLink KLH Conjugation Kit (AAT BIOQUEST, 5502) to couple the C-23 polypeptide and the recombinant Chemerin protein respectively to prepare a KLH-protein conjugate and purify the conjugate.

[0087] (2) The above-mentioned KLH-protein conjugates were used to immunize 6-8 week-old female Balb / c mice respectively. After immunization for 4 times, spleen cells of the mice were aseptically collected for cell fusion.

[0088] (3) Myeloma cells were revived 2 weeks before cell fusion, and mouse peritoneal macrophages were prepared and cultured 1-2 days before, and the obtained splenocytes and bone marrow cells were co-cultured in HAT medium containing peritoneal macrophages. Frequently observe the hybrid cell tumor, and when it grows to more than 1 / 10 of the area of ​​the bottom of the well, aspirate the supernatant for antibody detection.

[0089] (4) Enzyme-linked...

Embodiment 2

[0092] Preparation of the C-23 antibody labeled with colloidal gold in Example 2:

[0093] Preparation of colloidal gold using the Gold-in-a-box Conjugation Kit

[0094] (1) Vortex and resuspend the gold sol, put 0.5ml bare gold sol in 10 clean Ep tubes;

[0095] (2) Paste the pH 1-10 label on the Ep tube, use the pH value chart (Table 1), add different amounts of the buffer prepared in the kit to each test tube, and vortex to mix;

[0096] (3) Vortex each Ep tube at low speed, add 14 μl (2.5 mg / ml) of C-23 antibody solution, and place at room temperature for 30 minutes to complete the reaction;

[0097] (4) Add 50 μl of Blocking Stabilizer Soltion to stop the reaction, and stop the reaction at room temperature for 16 hours.

[0098] (5) In order to effectively dry the colloidal gold, add 0.05ml drying buffer and mix thoroughly. Coated on the colloidal gold film and incubated overnight at 37°C.

[0099] (6) After testing, the colloidal gold with pH=8.4 is relatively stable...

Embodiment 3

[0102] Embodiment 3 production process

[0103] (1) Preparation of sample pad: 0.5% ovalbumin, 0.01% teen-20, 0.02% sodium azide preservative, 0.01M PBS with pH7.0, make sample pad treatment solution, soak the sample pad in the sample After padding with the treatment solution, dry overnight at 37°C and store in an airtight seal at room temperature.

[0104] (2) Preparation of gold standard pad: use Gold-in-a-box Conjugation Kit (20nm) to prepare immunogold, pH value is 8.4, concentration is 56 μg / ml, dilute the immunogold to 50 μg / ml, And coated on the colloidal gold bonding pad, the dosage is about 0.24μg / cm 2 , dried at 37°C, and stored at 4°C for later use.

[0105] (3) Preparation of solid-phase nitrocellulose membrane (NC): 2% bovine serum albumin, 2% skimmed milk powder and 0.01M, pH7.4 PBS buffer were mixed, and the blocking solution was filtered with a 0.45 μm filter membrane. Dilute the mouse anti-human Chemerin antibody and anti-mouse secondary antibody with PBS b...

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PUM

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Abstract

The invention relates to a detection test strip for chemerin and a derivative polypeptide thereof and a preparation method of the detection test strip and in particular discloses a test strip for detecting chemerin. The detection test strip comprises a detection film, wherein a binding region containing a first antibody marked by a marker is arranged at the upstream of the detection film, a sample adding region is arranged at the upstream of the binding region, the detection film is provided with a detection region and a control region, and the control region is arranged at the downstream of the detection region; a second antibody coats the detection region, and a secondary antibody containing the first antibody coats the control region; and the first antibody and the second antibody can be respectively and specifically bound with chemerin. The detection test strip provided by the invention is convenient, quick and economic and can realize semi quantitation.

Description

technical field [0001] The invention relates to the field of medical testing, in particular to a chemokine detection test strip and a preparation method thereof. Background technique [0002] Chemerin is a newly discovered adipokine, which is mainly secreted by adipose tissue in the form of autocrine or paracrine, mainly expressed in white adipose tissue, liver and lung, and also expressed in adrenal gland, pancreas, placenta, ovary, skin, etc. . Chemerin can bind to its receptor, recruit inflammatory cells to participate in inflammatory response, regulate insulin sensitivity and adipocyte differentiation, thereby affecting glucose and lipid metabolism, and plays an important role in obesity and metabolic syndrome. In addition, chemerin has been reported to exhibit an inhibitory effect on gonadal hormone secretion, therefore, chemerin may also play a role in steroid synthesis and hormone secretion in reproductive diseases associated with obesity and metabolic abnormalities ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/558
CPCG01N33/558G01N33/6893G01N33/54387
Inventor 张键李梦霞陈杰肖天霞
Owner SHENZHEN INST OF ADVANCED TECH
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