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Neutrophil gelatinase-associated lipocalin detection kit and preparation

A lipocalin, neutrophil technology, applied in biological testing, measuring devices, material inspection products, etc., can solve problems such as unsatisfactory, inappropriate, false negatives, etc., achieve large market competitiveness and prolong service life , the effect of reducing manufacturing costs

Active Publication Date: 2014-08-20
BEIJING JIUJIAYI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The research of the present invention shows that many nanoparticles do not meet the requirements of the test within this range, such as: when the magnetic nanoparticles such as iron oxide are more than 40nm, they are easy to coagulate and settle due to factors such as paramagnetism and specific gravity, and cannot achieve proper stability; The stability of gold nanoparticles in the range of 80-150nm is too poor and unsuitable, etc.
According to the teaching of Chinese patent CN102749454, the present invention tests colloidal gold particles with a diameter of 35nm-60nm, and finds that the sensitivity is poor and cannot meet the clinical requirements for sensitivity less than 1ng / mL and the minimum detection limit of 5ng / mL , and the linear range does not cover the concentration range of normal physiological and pathological conditions in clinical practice, so false negative results are often caused; at the same time, there is also a long reaction time (equilibrium time 5-10min), which cannot meet the requirements of rapid testing

Method used

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  • Neutrophil gelatinase-associated lipocalin detection kit and preparation
  • Neutrophil gelatinase-associated lipocalin detection kit and preparation
  • Neutrophil gelatinase-associated lipocalin detection kit and preparation

Examples

Experimental program
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Effect test

Embodiment 1

[0036] The preparation of reagent R2 in this example is divided into three steps. Firstly, a colloidal gold solution is prepared, then the antibody is coupled to the gold nanoparticles, and finally it is mixed with buffer salt, coagulant and preservative to form a complete system.

[0037] The specific process is as follows:

[0038] 1) The preparation of colloidal gold of 50nm is as follows:

[0039] Put a magnetic stirrer in a cleaned 1000ml round bottom flask, and add 500ml of ultrapure water;

[0040] Add 5ml concentration and be 1% (w / v) chloroauric acid, stir at about 600rpm at a high speed, and be heated to solution boiling, be that 1% (w / v) sodium citrate solution is added in the flask rapidly then with 6ml concentration, keep Heating and stirring vigorously for 10 minutes, the color of the solution first turns black, and then gradually turns purple;

[0041] Turn off the heating switch and continue to stir for 10 minutes, then stop stirring and cool to room temperat...

Embodiment 2

[0060] The only difference between Example 2 and Example 1 is that the particle size of the colloidal gold particles prepared in Example 2 is 55 nm by adjusting the amount of reducing agent added.

[0061] Prepare 55nm particles and add reducing agent 1% sodium citrate to 5.6ml. The obtained colloidal gold particles were observed by transmission electron microscope, and the diameter was about 56.2±1.5nm.

Embodiment 3

[0063] The only difference between Example 3 and Example 1 is that the particle size of the colloidal gold particles prepared in Example 3 is 62.2 nm by adjusting the amount of reducing agent added.

[0064] Prepare 62.2nm particles and add reducing agent 1% sodium citrate to make 5.4ml. The obtained colloidal gold particles were observed by transmission electron microscope, and the diameter was about 62.2±1.1nm.

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Abstract

The invention provides a neutrophil gelatinase-associated lipocalin detection kit. Based on colloidal gold immunoturbidimetry, the kit contains a reagent R2 which is a solution containing gold nanoparticles labeled with a neutrophil gelatinase-associated lipocalin antibody. The kit is characterized in that particle size of the gold nanoparticles is 62.2nm-79.1nm; and the mass ratio of the gold nanoparticles to the antibody is 50:20-60. The invention also provides a preparation method of the kit. The kit provided by the invention has characteristics of high sensitivity, high specificity, rapid reaction and good stability. No precipitate is generated after a reaction. It is convenient to clean a biochemical analyzer, and service life of the biochemical analyzer is prolonged.

Description

technical field [0001] The present application relates to a neutrophil gelatinase-associated lipocalin detection kit based on colloidal gold immunoturbidimetry and its preparation. Background technique [0002] In 1993, neutrophil gelatinase-associated lipocalin (NGAL) was discovered in peroxidase granules in neutrophils. The full-length cDNA of NGAL is 5869bp, with 198 amino acids, including a signal peptide sequence consisting of 20 amino acids at the N-terminal and a peptide segment containing 178 amino acid residues. It is a secreted protein with a relative molecular mass of about 25KDa. The isoelectric point is 8.40. [0003] Under physiological conditions, a small amount of NGAL is synthesized in the middle and late stages of neutrophils in the bone marrow, and inflammatory reactions and malignant tumors can induce its production in multiple tissues (such as uterus, prostate, salivary glands, lungs, kidneys, airways and digestive tracts). Epithelial, etc.) in large q...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531
CPCG01N33/54346G01N33/92G01N2405/00
Inventor 侯淑霞王万霞
Owner BEIJING JIUJIAYI TECH
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