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Method for immunological detection for biological molecule of body fluid by gold magnetic particle

A technology for immunological detection and gold magnetic particles, applied in the field of immunological detection, can solve the problems of easily damaged conjugated biomolecules, long detection time, small fixed capacity, etc., to avoid matrix effects and to achieve fast detection. , the effect of large fixed capacity

Inactive Publication Date: 2008-04-23
SHANXI LIFEGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a method for immunological detection of biomolecules in body fluids using gold magnetic particles, which solves the problem of the long detection time and small fixed capacity, relatively low sensitivity, and coupled biomolecules in the background technology. The activity is vulnerable to technical problems

Method used

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  • Method for immunological detection for biological molecule of body fluid by gold magnetic particle
  • Method for immunological detection for biological molecule of body fluid by gold magnetic particle
  • Method for immunological detection for biological molecule of body fluid by gold magnetic particle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Immobilization of antibodies specific to surface antigen of hepatitis B virus.

[0048] (1) Pretreatment: take 200 μl of 5 mg / ml gold magnetic particles, and wash them twice with Tris-HCl coupling buffer of pH 7.4, each time 0.4 ml.

[0049] (2) Coupling: Dissolve 10 μg of anti-HBsAg monoclonal antibody to be coupled in 400 μl of coupling buffer, mix well, add to pretreated gold magnetic particles, place on a shaker, and store at 37°C The condition of 180rpm fully reacted for 20min. After the reaction is completed, magnetic separation is performed, and the supernatant is discarded.

[0050] (3) Equilibration: add 400 μl 1×PBS equilibration buffer, magnetically separate, and discard the supernatant.

[0051] (4) Blocking: add 1 ml of blocking solution, react in a shaker at 37° C. and 180 rpm for 2 hours, magnetically separate, and discard the supernatant. Wash 3 times with 3 ml of phosphate washing buffer plus 0.05% Tween 20, and finally suspend in 1 ml of ...

Embodiment 2

[0052] Example 2: Detection of Treponema pallidum antibodies in serum / plasma.

[0053] (1) Fixation and blocking: Take 100 μl, 5 mg / ml gold magnetic particles and place them on a magnetic separation rack, add 0.02 M, pH=7.4, 200 μl Tris-HCl coupling buffer to equilibrate for 2 minutes, and discard the supernatant. Add 400 μl Tris-HCl coupling buffer to wash the magnetic particles, magnetically separate, discard the supernatant, and repeat twice. Add TPN15, TPN17 and TPN47 three antigens, use Tris-HCl coupling buffer to make up to 200μl, place the centrifuge tube containing gold magnetic particles and antigens on a shaker, fully react at 37°C, 180rpm for 30min, and the reaction is complete , take it out and place it on a magnetic separation rack, discard the supernatant by magnetic separation, wash twice with 400 μl Tris-HCl equilibration buffer, and discard the supernatant. Add 1ml of blocking solution containing 20% ​​calf serum, 4% skimmed milk powder, dissolved in Tris-HCl...

Embodiment 3

[0055] Example 3: Detection of human interleukin-8 (IL-8) in serum / plasma.

[0056] (1) Pretreatment: the ascites fluid of the anti-IL-8 monoclonal antibody was preliminarily purified by 45% ammonium sulfate, and after dialysis, it was directly coated on the surface of gold magnetic particles. Take 0.4ml of gold magnetic particles, magnetically separate, discard the supernatant, and wash twice with 0.4ml of 0.02M, pH7.4 Tris-HCl coupling buffer to balance the pH of the magnetic particles.

[0057] (2) Coupling: Take 70 μg of antibody solution, dissolve it in Tris-HC coupling buffer, add it to the magnetic particles, and put it on a shaker for reaction at 170 rpm, 37° C., and 20 min.

[0058] (3) Equilibration: the antibody-coated magnetic particles were washed once with 0.01M Tris-HCl washing buffer containing 0.001M EDTA·2Na pH7.4, and 0.5×PBS equilibrating buffer.

[0059] (4) Sealing: Take 1ml of 5% skimmed milk powder dissolved in PBS and add it to the magnetic particles,...

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Abstract

The method comprises: using GoldMag nano-particles as the solid-phase carrier of reaction and separation; coupling the antibody or antigen to the surface of GoldMag nano-particles in order to make the antibody and antigen on the surface of GoldMag nano-particle to combine with the specific antibody or antigen in the serum so as to form an antibody-antigen composite; combining said antibody-antigen composite on the GoldMag nana-particles with the specific antibody or antigen marked by the marker; using relevant detection system to make qualitative or quantitative detection for the biomolecule under test.

Description

technical field [0001] The invention belongs to the field of immunological detection, and in particular relates to a method for immunological detection of biomolecules in body fluid by using gold magnetic particles as carriers. Background technique [0002] Most of the traditional methods for detecting biomolecules in body fluids use enzyme-linked immunoassays using polystyrene and other materials as carriers, which are relatively simple, but their disadvantages are poor detection sensitivity and long reaction time. [0003] Magnetic particles, also known as magnetic microspheres or magnetic beads, are a colloidal liquid formed by superparamagnetic nanoparticles, including magnetic metals such as Fe, Co, Ni or their metal oxides, and polymers or inorganic materials. The complex, in addition to the characteristics of nanoparticles, can also perform various surface functional modifications through the functional groups on the surface; moreover, it has paramagnetism and can mov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/553
Inventor 崔亚丽陈超付强
Owner SHANXI LIFEGEN
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