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Human cystatin C chemiluminescence quantitative detection method

A quantitative detection method and chemiluminescence technology, applied in chemiluminescence/bioluminescence, analysis by chemical reaction of materials, measurement devices, etc., can solve the problems of long detection time, complicated operation and low sensitivity.

Inactive Publication Date: 2013-04-03
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantages of latex-enhanced immunoturbidimetric method are good detection specificity, simple operation, high precision and wide detection range, but the disadvantage is low sensitivity
Enzyme-linked immunoassay and chemiluminescence (application number: 201010245894.2) are highly sensitive, but the operation is complicated, the detection takes a long time, the detection range is narrow, and the sample needs to be pre-diluted

Method used

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  • Human cystatin C chemiluminescence quantitative detection method
  • Human cystatin C chemiluminescence quantitative detection method
  • Human cystatin C chemiluminescence quantitative detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Anti-FITC antibody was coupled with magnetic particles to prepare a magnetic separation reagent.

[0015] Materials and Instruments

[0016] 1. Magnetic particles: containing carboxyl (COOH) active groups, the carboxyl content per gram (g) of magnetic particles (dry weight) is not less than 0.4 millimoles (mmol), with superparamagnetic properties, and the diameter is between 0.5-2 μm.

[0017] 2. Anti-FITC antibody: it can be polyclonal antibody or monoclonal antibody, the purity should be more than 90%, and the dilution titer should be more than 1:1 million.

[0018] 3.2-Morpholineethanesulfonic acid (MES), carbodiimide (EDC), TRIS and other reagents should be chemically pure.

[0019] Steps

[0020] 1. Take 100 mg of magnetic particles, remove the supernatant by magnetic separation, and resuspend in 10 ml of 0.05 mol / L, pH=4.5-5.0 MES buffer;

[0021] 2. Add 2-4mg of anti-FITC antibody and suspend at room temperature for 30-60min;

[0022] 3. Add 0.5-1m...

Embodiment 2

[0024] Example 2: Preparation of reagent 1 by linking FITC with anti-CYS-C antibody

[0025] Materials and Instruments

[0026] 1. Anti-human CYS-C monoclonal antibody, prepared by Beijing Leadman Biochemical Co., Ltd., with a purity of more than 95%, a concentration of more than 1mg / ml, and stored in phosphate buffer;

[0027] 2. Fluorescein isothiocyanate (FITC), sodium carbonate and other reagents should be chemically pure;

[0028] 3. The G-25 gel purification column was purchased from GE.

[0029] Steps

[0030] 1. Use 0.1-0.2 mol / L carbonate buffer solution with pH=9.0-10.0 to prepare 0.5mg / ml FITC solution;

[0031] 2. Add FITC solution to the anti-human CYS-C antibody solution according to the ratio of anti-human CYS-C antibody to FITC molar ratio of 1:20-1:200, mix well, and stand at room temperature for more than 12 hours;

[0032] 3. Use a G-25 gel column to separate the anti-human CYS-C antibody-FITC conjugate from free FITC to obtain a concentrated solution of...

Embodiment 3

[0033] Example 3: Preparation of Reagent 2 by linking ALP to anti-human CYS-C antibody

[0034] Materials and Instruments

[0035] 1. Anti-human CYS-C monoclonal antibody, prepared by Beijing Leadman Biochemical Co., Ltd., with a purity of more than 95%, a concentration of more than 5mg / ml, and stored in phosphate buffer;

[0036] 2. The purity of alkaline phosphatase should exceed 95%, the specific activity should exceed 1000 U / mg, and the concentration should exceed 5mg / ml;

[0037] 3. Coupling agent SMCC, 2-IT were purchased from THERMO company, TRIS and other chemical reagents should be chemically pure;

[0038] 4. AKTA-purifier protein purification instrument and Supperdex200 gel purification column are products of GE Company.

[0039] Steps

[0040] 1. Take 1 mg of anti-human CYS-C antibody, add 2-4 μl of 10 mg / ml coupling agent 2-IT solution, let it stand at room temperature for 20 minutes, add 10 μl of 0.1 mol / L glycine solution, and let it stand at room temperature...

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Abstract

The invention provides a human cystatin C quantitative detection method. According to the method, a magnetic micro-particle separation technology and an enzymatic chemiluminescence technology are combined, the principle of a double-antibody sandwich one-step reaction method is adopted, and the method can be used for content determination of human cystatin C in serum, plasma and urine samples and assistance in diagnosis and treatment of various renal dysfunction diseases. The sensitivity of the method is not more than 0.01mu g / L, the quantitative detection range is 0.05-8mu g / L, the precision in analysis is less than 5%, the detection time is 10 minutes, the recovery rate of the added serum and urine is 90-115%, and the correlation analysis R2 with a reagent serum value detected by a latex-enhanced immunoturbidimetric method is 0.9862.

Description

technical field [0001] The invention belongs to the field of biotechnology and provides an accurate, sensitive, rapid and quantitative chemiluminescent immunodetection method for cystatin C (CYS-C) in human serum, plasma and urine samples. Background technique [0002] Cystatin C is a non-glycosylated protein and a kind of cysteine ​​protease inhibitor. It consists of 122 amino acids and has a relative molecular weight of about 13kDa. It is produced in a constant manner by the majority of nucleated cells, filtered by the glomerulus, fully absorbed and broken down in the proximal convoluted tubule. Its molecular weight is small, and its production rate is stable. Since its concentration in serum is closely related to glomerular filtration rate (GFR), and it is more sensitive to glomerular filtration rate than creatinine, cystatin C has a great influence on Determination of glomerular filtration rate is of great significance. Cystatin C is completely reabsorbed by the proxim...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531G01N21/76
Inventor 不公告发明人
Owner BEIJING LEADMAN BIOCHEM
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