Neutrophile granulocyte gelatinase related lipid transport protein detection kit and preparation method thereof

A technology of lipocalin and neutrophils, which is applied in biological testing, measuring devices, material inspection products, etc., can solve problems such as poor anti-interference ability, low degree of automation of enzyme-linked immunoassay, and small linear range of measurement , to avoid the impact of product quality, solve the problem of insufficient product stability, and avoid the effect of poor product stability

Active Publication Date: 2013-04-17
WUHAN LIFE ORIGIN BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzyme-linked immunoassay has a low degree of automation and is greatly affected by human factors; radioimmunoassay has environmental pollution problems; Western-blotting is complicated to operate and has low measurement precision; while chemiluminescence method has high sensitivity, but its linear range is relatively narrow. Small size and high detection cost, requiring a specific chemiluminescence detector, these reasons lead to a small range of applications; latex immunoturbidimetry is also a common method for determining the concentration of NGAL in hu

Method used

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  • Neutrophile granulocyte gelatinase related lipid transport protein detection kit and preparation method thereof
  • Neutrophile granulocyte gelatinase related lipid transport protein detection kit and preparation method thereof
  • Neutrophile granulocyte gelatinase related lipid transport protein detection kit and preparation method thereof

Examples

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Example Embodiment

[0048] Preliminary Example 1 Screening of NGAL antibodies

[0049] 1. Preparation of MMP-9 affinity chromatography column: Mix protein MMP-9 (purchased from R&D company) with packed product CNBr-Sepharose4B (Pharmacia product) at a ratio of 5-7mg / mL, shake at 4°C for 20 hours, Make it crosslinked. Then wash with 0.1mol / L pH8.0 carbonate buffer for 3 times, and then add the same amount of 0.1mol / L ethanolamine-HCl 4°C and shake for 1 hour to block the remaining activated groups. Finally, use 0.01mol / L Tris-HCl (TB) to clean, pack, and equilibrate. The volume of the column is 10 mL.

[0050] 2. Preparation of MMP-9 / NGAL complex affinity chromatography column: The protein NGAL (purchased from R&D company) was prepared into a 2mg / mL solution, and the NGAL protein solution was passed through the MMP-9 affinity at a flow rate of 1mL / min. Chromatography column, the amount of protein solution is 60mL; then use 0.1mol / L pH8.0 phosphate buffer to elute the excess NGAL protein at a flow rat...

Example Embodiment

[0052] Preliminary Example 2 Preparation of latex particles coated with NGAL antibody

[0053] The preparation process of latex particles coated with NGAL antibody is as follows:

[0054] ①Preparation of carboxylated modified polystyrene latex solution: Weigh 1.0g dried polystyrene latex particles with a particle size of 105nm, add 100mL 0.12M phosphate buffer (pH 7.5) for dispersion, and add 40mg 1-ethyl -3-(3-Dimethylaminopropyl)carbodiimide (EDC), then add 20mg of thio-NHS, stir the reaction at room temperature for 0.5 hours, centrifuge to discard the supernatant, and then use 100mL 0.12M phosphate buffer Liquid dilution, dispersion and precipitation latex to obtain carboxylated modified polystyrene latex;

[0055] ② Preparation of NGAL antibody solution: Dissolve 250 μg of the NGAL antibody screened in Preliminary Example 1 in 0.5 mL of deionized water to obtain an NGAL antibody solution;

[0056] ③Preparation of latex particles coated with NGAL antibody: Take 100 μL of the NGAL ...

Example Embodiment

[0057] Preparatory Example 3 Preparation of latex particles coated with NGAL antibody

[0058] The preparation process of latex particles coated with NGAL antibody is as follows:

[0059] ①Preparation of carboxylated modified polystyrene latex solution: Weigh 1.0g of dried polystyrene latex particles with a particle size of 130nm, add 100mL 0.12M phosphate buffer (pH 7.5) for dispersion, and add 40mg 1-ethyl -3-(3-Dimethylaminopropyl)carbodiimide (EDC), then add 20mg of thio-NHS, stir the reaction at room temperature for 0.5 hours, centrifuge to discard the supernatant, and then use 100mL 0.12M phosphate buffer Liquid dilution, dispersion and precipitation latex to obtain carboxylated modified polystyrene latex;

[0060] ② Preparation of NGAL antibody solution: 250 μg of the NGAL antibody screened in Preliminary Example 1 was dissolved in 0.5 mL of deionized water to obtain an NGAL antibody solution;

[0061] ③Preparation of latex particles coated with NGAL antibody: Take 100 μL of t...

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Abstract

The invention discloses a neutrophile granulocyte gelatinase related lipid transport protein detection kit and a preparation method thereof. The detection kit is composed of a reagent I and a reagent II independent from each other; the reagent I comprises the components of biobuffer, surfactant, coagulant, preservative, stabilizer, blocker, chelating agent and water; and the reagent II comprises the components of rubber latex particle of enveloping neutrophile granulocyte gelatinase related lipid transport protein antibody, biobuffer, chelating agent, surfactant, preservative, suspending agent, sealing agent, stabilizer and water. According to the invention, the neutrophile granulocyte gelatinase related lipid transport protein antibody of the detection kit can not only be combined with the neutrophile granulocyte gelatinase related lipid transport protein antigen, but also be combined with the MMP-9/NGAL (matrix metallopeptidase-9/ (neutrophil gelatinase associated lipocalin) compound, thereby avoiding the influence of MMP-9/NGAL compound to the detecting result, and having the advantages of good specificity, high sensitivity, wide linear range, favorable stability and the like.

Description

technical field [0001] The invention relates to a lipocalin content detection kit, in particular to a detection kit for neutrophil gelatinase-associated lipocalin (NGAL) content in a human body and a preparation method thereof, and the present invention further relates to the detection kit The method for detecting NGAL content in a human body belongs to the field of detection of NGAL content in a human body. Background technique [0002] Recent studies have found that a new member of the lipocalin family, neutrophil gelatinase-associated lipocalin (NGAL), is associated with acute kidney injury (AKI) and various It is closely related to chronic kidney disease (CKD) caused by various causes, and is a new marker for predicting AKI, as well as a biological indicator for monitoring the progress of CKD and renal function damage. [0003] NGAL is a member of the lipocalin family. It was discovered and isolated in human neutrophils by Kjeldsen et al. in 1993. Its rat and mouse inte...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
Inventor 华权高沈鹤霄黄爱许可马峰闫彩霞常向博舒芹
Owner WUHAN LIFE ORIGIN BIOTECH LTD
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