Detection kit of latex particle enhanced neutrophil gelatinase-associated lipid transfer protein

A lipocalin and neutrophil technology, applied in the biological field, can solve the problems of low sensitivity and accuracy, limited NGAL concentration, etc.

Active Publication Date: 2011-11-23
WUHAN LIFE ORIGIN BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the concentration of NGAL in urine or plasma is limited, which leads to the low sensitivity and accuracy of the traditional immunoturbidimetric method.

Method used

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  • Detection kit of latex particle enhanced neutrophil gelatinase-associated lipid transfer protein
  • Detection kit of latex particle enhanced neutrophil gelatinase-associated lipid transfer protein
  • Detection kit of latex particle enhanced neutrophil gelatinase-associated lipid transfer protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] A preparation method of a latex particle-enhanced neutrophil gelatinase-associated lipocalin detection kit.

[0013] 1. The preparation of reagent R1 of the present invention

[0014] Reagent R1 was 100 mM Tris buffer (pH 7.2), and Tritonx100 and PEG6000 were added to the buffer to a final concentration of 0.5%.

[0015] 2. The preparation of reagent R2 of the present invention

[0016] The linkage of antibody and latex was performed by chemical cross-linking. Proceed as follows:

[0017] (1) EDC and sulfo-NHS were activated for 15 minutes (PB Buffer), shaken, and centrifuged at 20,000 rpm for 30 minutes;

[0018] (2) After discarding the supernatant, redissolve with PB, add antibody and shake at room temperature for 2 hours;

[0019] (3) Add an equal amount of 0.1M glycine buffer solution with pH=8.0 and 10% BSA to make the final concentration 1%, react for half an hour, and centrifuge at 0000rpm for 30min;

[0020] (4) Reconstitute with the buffer of R1 reagent. ...

Embodiment 2

[0021] Embodiment 2 working curve is measured

[0022] The dosage of the present invention: the dosage of the reagents R1 and R2 of the present invention is 200 μl and 50 μl respectively, and the dosage of the sample is 3 μl.

[0023] The present invention adopts the endpoint method for measurement: 200 μl R1 is added to 3 μl sample, and 50 μl reagent R2 is added after 5 minutes at 37°C to start reading the point, and then read the point again after 5 minutes, and then calculate the absorbance to calculate the difference. The kit was determined using Mindray BS300 automatic biochemical analyzer, the main detection wavelength was 546nm, and the secondary wavelength was 670nm.

[0024] Make the standard curve of the present invention: adopt standard substance of the present invention (choose 6 points to calibrate, calibrator NGAL content is respectively 0.0,0.05,0.1,0.2,0.4,0.8mg / ml), records NGAL of the present invention according to above-mentioned measuring procedure The sta...

Embodiment 3

[0025] Embodiment 3 accuracy measurement

[0026] Correlation experiment: using the reagent of the present invention and a control reagent (NGAL kit of a well-known international company). The mindray BS300 automatic biochemical analyzer was used to measure 30 human sera, and the correlation analysis was carried out on the measured values. See the test results figure 2 , the X and Y axes in the figure are measured values ​​(NGAL content mg / L). The results show that the correlation between the present invention and the control reagent is high.

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Abstract

The invention provides a detection kit of a latex particle enhanced neutrophil gelatinase-associated lipid transfer protein and a preparation method thereof. The content of the neutrophil gelatinase-associated lipid transfer protein in urines, serums or blood plasmas of humans can be detected by the kit of the invention. Diameters of latex particles of the latex particle enhanced neutrophil gelatinase-associated lipid transfer protein are from 30nm to 220 nm. The kit of the invention has the advantages of good specificity, high sensitivity, strong anti-interference capability and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a latex particle-enhanced neutrophil gelatinase-associated lipocalin detection kit. Background technique [0002] Neutrophil gelatinase-associated lipocalin (NGAL) was first discovered in neutrophils in 1993. It is also called lipocalin2 and is a new member of the lipocalin family. Its structure and function have attracted attention at home and abroad. . NGAL protein is widely distributed in human tissue cells, such as bronchi, stomach, small intestine, pancreas, kidney, prostate, thymus, etc. NGAL protein has the functions of transporting small hydrophobic molecules, regulating the activity of gelatinase B (MMP-9), and may participate in the immune inflammatory response and the occurrence and development of tumors, especially the process of tumor invasion and metastasis. Studies have shown that NGAL gene may be a new human oncogene or tumor-promoting gene. [0003] Usually, more th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/566G01N21/31
Inventor 华权高沈鹤霄周许可
Owner WUHAN LIFE ORIGIN BIOTECH LTD
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