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C-reactive protein chemiluminiscence immunoassay kit and preparation method and application thereof

A technology of chemiluminescence immunoassay and detection kit, which is applied in the fields of chemiluminescence/bioluminescence, biological testing, and analysis through chemical reactions of materials, etc., and can solve problems such as limitations, narrow linear range, and rare chemiluminescence immunoassays, etc. problem, to achieve accurate detection, improve linear range and sensitivity

Inactive Publication Date: 2020-12-08
WUHAN LIFE ORIGIN BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The methods for detecting C-reactive protein in the prior art mainly include immunoturbidimetry, fluorescence immunochromatography, enzyme-linked immunoassay and chemiluminescence immunoassay; the common methodology for detecting C-reactive protein on the market is immunoturbidimetry, Chemiluminescence immunoassay is relatively rare, mainly because: although chemiluminescence immunoassay has higher sensitivity than immunoturbidimetry, its linear range is narrow, and the content of C-reactive protein in human body is relatively high, which leads to chemiluminescence Immunoassays have limited use in clinical testing of C-reactive protein

Method used

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  • C-reactive protein chemiluminiscence immunoassay kit and preparation method and application thereof
  • C-reactive protein chemiluminiscence immunoassay kit and preparation method and application thereof
  • C-reactive protein chemiluminiscence immunoassay kit and preparation method and application thereof

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Embodiment 1

[0036] Embodiment 1 reagent preparation

[0037] (1) Magnetic bead cleaning solution: pH 5.0, including: 100mM MES buffer, 0.05% Tween20;

[0038](2) Magnetic bead activator: EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (1-(3-di Methylaminopropyl)-3-ethylcarbodiimide hydrochloride) solution;

[0039] (3) Coupling buffer: a 100mM boric acid solution with a pH of 8.0-8.5;

[0040] (4) Blocking buffer: pH 7.2, including 50-100mM PBS buffer, 1% bovine serum albumin (BSA);

[0041] (5) Preservation solution: 100mM Tris buffer, 0.1% preservative PC-300;

[0042] (6) Antibody activator: 100mM Tris buffer, 0.3% NaCl, 5mM EDTA, 10mg / mL 2-IT (2-iminothiophene) cross-linking agent;

[0043] (7) Enzyme activator: 100mM Tris buffer, 0.3% NaCl, 5mM EDTA, 5mg / mL SMCC cross-linking agent dissolved with dimethylformamide;

[0044] (8) M dilution: pH 7.2, including: 25mM HEPPSO buffer, 0.5% Tween20, 1% BSA, 1% pullulan, 0.1% PC-300;

[0045] (9) R diluent: pH ...

Embodiment 2

[0047] The preparation of embodiment 2 kit

[0048] (1) C-reactive protein antigen: purity>95%, purchased from Wuhan Huamei Bioengineering Co., Ltd.

[0049] (2) C-reactive protein antibody: four kinds of mouse anti-human C-reactive protein monoclonal antibodies with different affinities, namely a, b, c, d, with a purity of >95%, were purchased from Wuhan Huamei Bioengineering Co., Ltd., and the article numbers are respectively : CSB-DA402GmN②(a), CSB-DA402GmN③(b), CSB-DA402GmN⑤(c), CSB-DA402GmN⑥(d), the affinity constants of the four antibodies are shown in the following table:

[0050] C-reactive protein antibody Affinity constant Kd(mol / L) a 1.56×10 -12

b 2.47×10 -10

c 5.26×10 -6

d 3.58×10 -7

[0051] Wherein the order of magnitude of the affinity constant Kd of antibody a and b is 10 -9 ~1×10 -12 between, indicating that the antibody has a high affinity, and the affinity constant Kd of antibodies c and d is on the order of...

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Abstract

The invention discloses a C-reactive protein chemiluminiscence immunoassay kit and a preparation method and application thereof. The C-reactive protein chemiluminiscence immunoassay kit comprises an Mreagent, an R reagent, a luminescent substrate solution and a calibrator, the M reagent is a magnetic bead reagent coated with an antibody M, the R reagent is an enzyme labeling reagent connected with an antibody R, and both the antibody M and the antibody R can be specifically combined with C-reactive protein; the ratio of the affinity constants of the antibody M and the antibody R is 1 * 10<-7>to 1 * 10<-2> or 1 * 10<2> to 1 * 10<7>. By changing the affinity of the antibody M coated by the magnetic beads and the antibody R labeled by the enzyme, the affinity constants of the antibody M andthe antibody R are remarkably different, so that the linear range and sensitivity of the C-reactive protein chemiluminescence immunoassay kit are improved, and the problems of low sensitivity and high sensitivity of the traditional immunoturbidimetry, and the chemiluminescence immunoassay is narrow in linear range, so that accurate detection of the C-reactive protein is achieved in clinical use.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnostic reagents for immunological detection, and in particular relates to a C-reactive protein chemiluminescence immunological detection kit and a preparation method and application thereof. Background technique [0002] C reactive protein (C reactive protein, CRP) is synthesized by liver cells, produced in the fetus, non-maternal placental transmission. Its production mechanism is: when the body is infected or the tissue is damaged, macrophages and other white blood cells are activated to produce interleukin-6, interleukin-1, tumor necrosis factor TNF-a and other cytokines and other mediators. These cytokines and mediators reach the liver and stimulate hepatocytes and epithelial cells to synthesize CRP. Structurally, CRP contains 5 polypeptide chain subunits, which are non-covalently combined into a disc-shaped polymer with a molecular weight of 115,000 to 140,000. CRP is a typical acute ph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/543G01N21/76
CPCG01N33/6893G01N33/577G01N33/54326G01N21/76G01N2333/4737G01N2800/324
Inventor 潘鑫来祥兵张雪娇舒芹
Owner WUHAN LIFE ORIGIN BIOTECH LTD
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