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Quantum point marker sandwich immunodetection method and its diagnosis kit

A technology of sandwich immunity and quantum dots, applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of unstable color development, long detection cycle, cumbersome operation, etc., and achieve simple and easy operation, strong The effect of resistance

Inactive Publication Date: 2004-07-28
魏景艳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the operation is cumbersome, time-consuming, the detection cycle is long, and the color development is unstable

Method used

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  • Quantum point marker sandwich immunodetection method and its diagnosis kit
  • Quantum point marker sandwich immunodetection method and its diagnosis kit
  • Quantum point marker sandwich immunodetection method and its diagnosis kit

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Quantum dot-labeled sandwich immunoassay of cardiac troponin I (cTnI)

[0040] (1) Anti-cTnI monoclonal antibody was labeled with red light-emitting QD630 to prepare detection antibody

[0041]Method 1: Relying on electrostatic attraction connection: the biomolecules are connected to a layer of negatively charged free radicals coated on the surface of QDs. The methods include: a) Coating a layer of dihydrolipoic acid (DHLA) on the outer layer of QDs, and then connecting with avidin, and then biotinylated proteins, nucleic acids, and even cell membranes as needed, relying on biotin-affinity The highly specific binding force between proteins will label QDs to the target molecule; b) directly connect the positively charged protein to the QDs; c) connect the positively charged protein to the zipper through a positively charged leucine zipper protein as a bridge The mAb at the other end is labeled with QDs.

[0042] Method 2: Covalent coupling: a) QDs are coated with a lay...

Embodiment 2

[0057] Specificity of a Quantum Dot-Labeled Sandwich Immunoassay for Cardiac Troponin I (cTnI) (Validation of Method Specificity)

[0058] In order to illustrate the specificity of the sandwich immunoassay results, BSA was used instead of cTnI as the antigen to be tested for immunoassay, and the whole process of Example 1 was repeated. In Example 2, the anti-cTnI monoclonal or polyclonal antibody and BSA are unpaired antigen antibodies or non-related antibodies, which do not have selective recognition, so they cannot specifically bind, nor can they form a three-layer sandwich luminescent immune system. Complex, that is, no fluorescence can be detected, indicating that the sample to be tested does not contain cTnI. The experimental results show that non-related proteins in the sample will not cause non-specific reactions, and the sandwich immunoassay is highly specific.

Embodiment 3

[0060] Quantum dot-labeled sandwich immunoassay for hepatitis B surface antigen (HBsAg)

[0061] Same as the steps in Example 1, the difference is that the capture antibody and detection antibody are anti-HBsAg monoclonal or polyclonal antibodies, and the antigen to be tested can only be HBsAg; the quantum dots used are QD570 with yellow light, fluorescent enzyme label The excitation light wavelength of the instrument is 460nm, the emission light is yellow, and the emission wavelength is 570±10nm. Similarly, the specimen containing HBsAg is used as the sample to be tested, and the concentration of HBsAg in the sample can be obtained by directly displaying or comparing with the standard curve by detecting the fluorescence intensity, see Figure 4.

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PUM

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Abstract

The present invention discloses a quantum point labeled sandwich immunodetection method and its diagnosis kit. It is a new type sandwich immunodetection method using QDs nano particle as label to make antigen antibody specificity sandwich reaction. It includes the following processes: firstly, directly or indirectly enveloping captured antibody in microwell of polystyrene plate, forming captured antibody-antigen-detection antibody three-layer sandwich luminescent immune complex and fluorescence intensity detection. According to that every QD has narrow and symmetrical fluorescence spectral peak it can select and use quantum point label needle sending different light to simultaneously detect several antigens to be tested in same sample.

Description

Technical field: [0001] The invention relates to a method for quantitatively detecting corresponding antigens with known antibodies labeled with quantum dots, and in particular discloses a quantum dot-labeled sandwich immunoassay and its diagnostic kit, which belong to the technical field of immunoassay methods. Background technique: [0002] The prior art close to the present invention is double-antibody sandwich enzyme-linked immunoassay (ELISA) or immunoenzyme quantitative assay (IEMA). See Larue C, et al. Clin. Chem. 1993, 39: 972-979. Cardiac-specific immunoenzymometric assay of troponin I in the early phase of acute myocardial infarction and other articles. This type of method uses a polyclonal or monoclonal antibody against the antigen to be tested as a capture antibody (primary antibody), another monoclonal antibody labeled with horseradish peroxidase or alkaline phosphatase as a detection antibody, and finally The substrate of the enzyme is added, and the color cha...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/535G01N33/573G01N33/577
Inventor 魏景艳房学迅李善玉杨柏王丽萍
Owner 魏景艳
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