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Sandwich immune detecting method

A detection method, sandwich immunology technology, applied in the field of biomolecular detection, can solve the problems of unstable color development, long detection cycle, low sensitivity, etc., and achieve the effect of low fluorescence background, strong resistance, and high photobleaching threshold

Inactive Publication Date: 2009-03-18
CHANGCHUN INST OF OPTICS FINE MECHANICS & PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the above-mentioned double-antibody sandwich enzyme-linked immunoassay, the marker used is enzyme, and the measured signal is absorbance, so it has the following disadvantages: cumbersome operation, time-consuming, long detection cycle, unstable color development, and low sensitivity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Sandwich immunoassay of UCP nanocrystal-labeled hepatitis B surface antigen (HBsAg)

[0023] 1. Yb, Er co-doped UCP nanocrystals labeled anti-HbsAg polyclonal antibody to prepare detection antibody.

[0024] Method 1: the surface is carboxyl Er, Yb co-doped nanocrystal-labeled anti-HbsAg polyclonal antibody, and the detection antibody is prepared;

[0025] Take the concentration as 10 -7 M UCP nanocrystal solution 2mL, respectively add 20μL 0.05M NHS and 20μL 0.5M EDC solution to UCP nanocrystal solution, stir for 30-60 minutes; then add 10 -5 M HbsAg polyclonal antibody 20μl.

[0026] Method 2: Nanocrystalline labeled anti-HbsAg polyclonal antibody with Er and Yb co-doped on the surface to prepare detection antibody;

[0027] Take the concentration as 10 -7 M UCP nanocrystal solution 2mL, add 20μL 0.05M glutaraldehyde solution to UCP nanocrystal, stir for 30-60 minutes; then add 10 -5 M HbsAg polyclonal antibody 20μl.

[0028] 2. Coating of capture antibody.

[...

Embodiment 2

[0039] Specificity of a UCP nanocrystal-labeled sandwich immunoassay for HbsAg.

[0040] In order to illustrate the specificity of the sandwich immunoassay results, BSA was used instead of HbsAg as the antigen to be tested for immunoassay, and the whole process of Example 1 was repeated. In Example 2, the anti-HbsAg monoclonal antibody and BSA used 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 immune complex, namely Fluorescence cannot be detected, indicating that the sample to be tested does not contain HbsAg. 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

[0042] Sandwich immunodetection of UCP nanocrystal labeling of AFP.

[0043] As shown in the steps of Example 1, the difference is that the capture antibody and detection antibody are anti-AFP monoclonal or polyclonal antibodies, and the antigen to be tested can only be AFP. By detecting the fluorescence intensity, it can be directly displayed or compared with the standard curve to obtain the concentration of AFP in the sample.

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Abstract

The invention relates to a biological molecule test method, in particular to a sandwich immunity test method for using known antibody to quantitatively test relative antigens, which comprises: directly or indirectly enveloping the capture antibody at the pores of a polystyrene plate, to form a sandwich luminous immunity compound; using a fluorometer to excite and detect the fluorescent intensity of the sandwich luminous immunity compound comprising three layers as the capture antibody, the antigen and the detection antibody; comparing the intensity with a standard solution to find the density of the object antigen; wherein the detection antibody is a single clone antibody labeled by upper conversion nanometer crystals and resisting the object antigen; and the different antigens in one sample can be tested by selecting the antibodies marked by the upper conversion nanometer crystals emitting different lights and resisting different antigens. The sandwich immunity test method has narrow fluorescent spectrum peak, adjustment emission spectrum, strong resistance for the degradation of chemicals and metabolism, high photonic bleaching value and low fluorescent background brightness. The sandwich immunity test method overcomes the defects of the prior sandwich immunity test methods, such as single color development and the limitation in the synchronous test of different objects.

Description

technical field [0001] The invention relates to a biomolecular detection method, in particular to a sandwich immunological detection method which uses known antibodies to quantitatively detect corresponding antigens. Background technique [0002] The traditional immunoassay method is double-antibody sandwich enzyme-linked immunoassay (ELISA). This type of method uses a polyclonal or monoclonal antibody against the antigen to be tested as a capture antibody, another monoclonal antibody labeled with horseradish peroxidase or alkaline phosphatase as a detection antibody, and finally adds an enzyme substrate , and calculate the type and concentration of the antigen to be tested by detecting the color change caused by the enzyme acting on the substrate with a microplate reader. The sensitivity of this method ranges from 0.2 to 1 μg / L. [0003] In the above-mentioned double-antibody sandwich enzyme-linked immunoassay, the marker used is enzyme, and the measured signal is absorba...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/545
Inventor 孔祥贵张友林曾庆辉孙雅娟刘晓敏
Owner CHANGCHUN INST OF OPTICS FINE MECHANICS & PHYSICS CHINESE ACAD OF SCI