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A kit for detecting the activity of dipeptidyl peptidase ⅳ and its application

A dipeptidyl peptidase and kit technology, applied in the field of biomedicine, can solve the problems of highly toxic substances and human body harm, and achieve the effect of high sensitivity

Active Publication Date: 2016-06-01
南京博炫生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under alkaline conditions, Gly-Pro-pNA is used as the enzyme substrate, and free p-nitroaniline and Gly-Pro (glycine-proline) dipeptide are generated after hydrolysis, using the chemiluminescence of p-nitroaniline The enzyme activity can be calculated by dynamically detecting the absorbance at 405nm. This method is simple to detect, but the disadvantage is that the toxicity of p-nitroaniline is a highly toxic substance, and it is harmful to the human body if inhaled or in contact with the skin.
Promega has introduced a detection method based on fluorescent luminophore, which has higher sensitivity and faster rate, but the detection requires a special fluorescence detector, and the operation needs to avoid fluorescence quenching

Method used

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  • A kit for detecting the activity of dipeptidyl peptidase ⅳ and its application
  • A kit for detecting the activity of dipeptidyl peptidase ⅳ and its application
  • A kit for detecting the activity of dipeptidyl peptidase ⅳ and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Example 1: Preparation of a kit for detecting dipeptidyl peptidase IV activity

[0017] A kit for detecting the activity of dipeptidyl peptidase IV, including liquid A and liquid B. The liquid A is prepared by dissolving Gly-Pro-X polypeptide powder in PIPES buffer, and the liquid B is prepared by dissolving tetranitrogen Prepared by dissolving azole salt, PMS and NaCl in PIPES buffer.

[0018] 1mol / LPIPES buffer configuration: Weigh PIPES (piperazine-N,N-bis(2-ethanesulfonic acid)) free acid according to the ratio, dissolve it in deionized water, adjust the pH to 6.9 with NaOH, add deionized water Dilute to 1mol / L, sterilize with a microporous filter, and store at 4°C for later use;

[0019] Configuration of solution A (enzyme reaction substrate solution): Dissolve glycine and proline polypeptide Gly-Pro-X powder (purchased by Gly-Pro-AMC Aimeijie Technology Co., Ltd.) in 1mol / LPIPES buffer, and prepare The final concentration was 50mmol / L, sterilized by microporous ...

Embodiment 2

[0023] Example 2: Application of the kit for detecting the activity of dipeptidyl peptidase IV

[0024] 1. Cultivate 1ml of Escherichia coli that can secrete and produce dipeptidyl peptidase IV in LB medium to the logarithmic culture period, collect the bacterial cells by centrifugation, add phosphate buffer to make 1ml of bacterial suspension, ultrasonically lyse for 10min, and centrifuge at 10,000rpm 30 minutes to collect the supernatant of ultrasonic lysis, as the sample to be tested;

[0025] 2. Add 100 μl of each well of the sample to be tested into a 96-well enzyme-labeled detection plate, and set up three replicate wells;

[0026] 3. Then add 10 μl of solution A and 10 μl of solution B to the well to be tested, and set the blank control as 100 μl of phosphate buffer solution, add 10 μl of solution A and 10 μl of solution B, and react at 37°C for 10 minutes;

[0027] 4. As an experimental control comparison, use the dipeptidyl peptidase IV detection reagent (PromegaDPPI...

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Abstract

The invention discloses a kit for detecting activity of dipeptidyl peptidase IV. The kit comprises a solution A and a solution B, wherein the solution A is prepared by dissolving Gly-Pro-X polypeptide powder in a PIPES buffer solution; the solution B is prepared by dissolving methyl thiazolyl tetrazolium, PMS and NaCl in the PIPES buffer solution. The invention also discloses application of the kit for detecting the activity of the dipeptidyl peptidase IV in detection of the activity of the dipeptidyl peptidase IV. According to unique reaction sensitivity of water-soluble methyl thiazolyl tetrazolium and non-toxicity of the reaction product, the water-soluble methyl thiazolyl tetrazolium serves as an indicator for detecting the activity of the dipeptidyl peptidase IV, so that a high-sensitivity detection range can be achieved, and pollution to experimenters and the environment is avoided.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a kit for detecting the activity of dipeptidyl peptidase IV and its application. Background technique [0002] Dipeptidyl peptidase IV can mediate the signaling pathway between T lymphocytes and antigen-presenting cells in eukaryotes, such as the signal transduction process involved in the pathological process of type 2 diabetes, and has important biological functions in the immune response At present, it has become a target molecule in the treatment of immune diseases and a new diagnostic index for disease treatment; in addition, it is widely present in various bacterial species, and as a virulence-related factor of various bacterial pathogenicity, it is an important factor for disease prevention and vaccine preparation. provides a new direction. With the in-depth research on dipeptidyl peptidase Ⅳ, its activity detection method has also been continuously improv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/78G01N21/31
Inventor 李忠马飞飞顾玉龙钱金蓉
Owner 南京博炫生物科技有限公司