Kit for detecting NGAL content and preparation method thereof

A kit and reagent technology are applied in the field of kits for detecting NGAL content and the field of preparation thereof, which can solve the problem that immune enhancement turbidimetry and latex immune turbidimetry have poor specificity, results are easily affected by subjective factors, and are easily affected by lipidemia. It can achieve the effect of no radioactive contamination, high sensitivity and strong specificity.

Inactive Publication Date: 2015-08-19
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the colloidal gold method has low sensitivity and is prone to false positives, and its results are easily affected by subjective factors; the immunoenhanced turbidimetric metho...

Method used

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  • Kit for detecting NGAL content and preparation method thereof
  • Kit for detecting NGAL content and preparation method thereof
  • Kit for detecting NGAL content and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The preparation method of embodiment 1NGAL antibody-coated magnetic microspheres

[0039] 1. Antibody pretreatment:

[0040] (1) Add 0.1mg of antibody (NGAL-coated antibody) into an ultrafiltration centrifuge tube (50kDa), and centrifuge at 8000r / min for 5-6min. Add 200uL Binding Buffer to the antibody concentrated by centrifugation, centrifuge at 8000-9000r / min for 5-6min, discard the filtrate, then add 200uL Binding Buffer, centrifuge as above, repeat this step 5-6 times.

[0041] (2) Take out the centrifuge tube after several times of centrifugation, discard the filtrate, invert the spin column, centrifuge at 2000-3000r / min for 1min, collect the filtrate, and collect about 200 μL of antibody during the whole process.

[0042] 2. Processing of magnetic microspheres:

[0043] (1) Use a vortex mixer to fully mix and suspend the magnetic microspheres, and take 4.0 mg of the suspension into a micro-volume thick-walled reaction bottle;

[0044] (2) Place the micro thick...

Embodiment 2

[0061] Example 2 Preparation method of acridinium ester-labeled NGAL antibody

[0062] 1. Antibody pretreatment:

[0063] (1) Add 0.13mg of the antibody into an ultrafiltration centrifuge tube (50kDa), centrifuge at 8000r / min for 5-6min to concentrate. Add the concentrated antibody to 200uL PB Buffer, centrifuge at 8000-9000r / min for 5-6min, discard the filtrate, then add 200uL PB Buffer, centrifuge as above, repeat this step 5-6 times.

[0064] (2) Take out the centrifuge tube after several times of centrifugation, discard the filtrate, invert the spin column, centrifuge at 2000-3000r / min for 1min, collect the filtrate, and collect about 200 μL of antibody during the whole process.

[0065] Antibody labeling: Mix the pretreated antibody and acridinium ester salt at a mass ratio of 50:1, and shake for 16-20 hours. Then the obtained product is purified by a purifier. Dilute the antibody labeled with acridinium ester salt to the final concentration with R2 diluent, and store ...

Embodiment 3

[0066] The usage method of embodiment 3 kit

[0067] 1. Loading Reagents

[0068] The 1.1M reagent is yellow-brown magnetic microsphere suspension, and precipitation is a normal phenomenon. Before loading the NGAL quantitative determination reagent on the Caris200 system for the first time, the M reagent bottle needs to be inverted and mixed.

[0069] 1.2 If the magnetic microspheres are still attached to the bottle wall by visual inspection, it is necessary to continue to flip until the magnetic microspheres are resuspended. The reagent cannot be used if it has not been suspended for a long time.

[0070] 1.3 Load the NGAL reagents on the Caris200 according to the requirements of the instrument to ensure that all the reagents required for the detection are loaded on the machine. Make sure all reagent bottle caps are removed.

[0071] 2. Calculate the required sample size

[0072] 2.1 When the NGAL test is performed for the first time, the sample needs to be diluted 1:10 ...

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Abstract

The invention relates to a kit for detecting neutrophil gelatinase-associated lipocalin content based on chemiluminescence immunoassay. According to the invention, by employing a double-antibody sandwich immunization analysis method, a chemiluminescence magnetic microspheres immunization technology is used, anti-NGAL antibody-coated magnetic microspheres for specifically combining with NGAL antigen of a standard substance/sample in a reaction cup, then are reacted to another strain anti-NGAL antibody labelled with acridine salt to form an immunization compound, through an acid-base chemical reaction of a pre-Trigger and a Trigger, relative light unit (RLU/s) of the chemiluminescence reaction can be measured; the NGAL antigen content in the sample is in direct proportion to the relative light unit (RLU/s) measured by an optical system, determination of NGAL content in an urine specimen can be determined through standard curve fitting; and the method has the obvious advantages of high sensitivity, strong specificity, good stability, simple operation and low cost.

Description

technical field [0001] The invention relates to a kit for detecting NGAL content and a preparation method thereof, and relates to a kit for detecting the concentration level of NGAL antigen in human urine by a double-antibody sandwich acridinium ester labeled tubular chemiluminescence immunoassay (Chemiluminesent Immunoassay, CLIA). Preparation Process. Background technique [0002] Acute kidney injury (acute kidney injury, AKI) is a clinical syndrome caused by a sudden decline in renal function in a short period of time (hours to days) caused by various reasons, and is a common disease that threatens the lives of critically ill patients. About 50% of patients in the intensive care unit can have this disease. At present, finding specific, sensitive, and stable early diagnostic markers, so as to achieve early diagnosis and early prevention of AKI, has become the key to reducing the mortality of severe patients. Traditional diagnostic indicators (such as urea nitrogen, serum...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 李明吴英松董志宁邓传欢黄小燕王红翠李志雄
Owner GUANGZHOU DARUI BIOTECH
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