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Construction Method of Antibody Microarray Based on Double Antibody Sandwich Immunoassay

A double-antibody sandwich and immunoassay technology, which is applied to the analysis of materials, material stimulation analysis, material inspection products, etc.

Inactive Publication Date: 2012-02-01
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In short, the current protein microarray technology is not yet mature, and its construction still needs further development and improvement.

Method used

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  • Construction Method of Antibody Microarray Based on Double Antibody Sandwich Immunoassay
  • Construction Method of Antibody Microarray Based on Double Antibody Sandwich Immunoassay
  • Construction Method of Antibody Microarray Based on Double Antibody Sandwich Immunoassay

Examples

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Embodiment 1

[0016] Example 1: Taking MCP-1 as an example, constructing a double-antibody sandwich antibody microarray: 1. Preparation of the carrier: Prepare a 1% agarose solution with a mass ratio of ultrapure water, and boil it in a microwave until it is completely dissolved; cover with 2ml of the agarose solution Put it on a preheated clean glass slide, and after the agarose solution solidifies at room temperature, put it in a 37°C oven to dry; 2. Activation of the carrier: immerse the carrier in 20mmol / L NaIO 4 The solution was activated for 30min, washed with 0.1M pH7.4 PBS for 5min * 3 times, sample immediately after drying with nitrogen flow; 3. Spotting and fixing of capture antibody (attached figure 1 Step 1): Spotting buffer preparation: 0.1mol / L Na 2 HPO 4 Solution, pH 9.0, containing glycerol 200ml / L; capture antibody diluted to 125ug / ml, 62.5ug / ml, 31.25ug / ml, and set positive control (streptavidin-cy3, biomarker detection antibody) and negative Control (200ug / ml BSA), us...

Embodiment 2

[0017]Example 2: Preparation of carrier: Prepare 0.5-1.5% agarose solution with ultrapure water, heat and boil until completely dissolved, cover the agarose solution on a preheated clean glass slide, solidify at room temperature, and dry in an oven at 37°C , placed at room temperature for subsequent use; carrier activation: the carrier made by step a is immersed in NaIO with a concentration of 20mmol / L 4 Activate in the solution for 20-30min, wash with 0.1M pH7.4 PBS, dry the activated carrier with nitrogen flow; spotting and fixing the capture antibody: first prepare 0.1mol / L Na with pH 9.0 and 200ml / L glycerol 2 HPO 4 Spotting buffer, the capture antibody is diluted with the spotting buffer according to the optimal titer concentration and added to the wells of the spotting plate, and then a positive control (streptavidin-cy3, biomarker detection antibody) is set on the wells of the spotting plate and Negative control (200ug / ml BSA) spotting, as microarray direction indicato...

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Abstract

A method for constructing an antibody microarray based on a double-antibody sandwich immunoassay, characterized in that the construction steps include: preparation of a carrier; activation of the carrier; spotting and fixing of a capture antibody; and immunoassay based on the microarray. The antibody microarray constructed according to this optimized operation procedure has good reproducibility (intra-group and inter-group variation <10%) and quantitative analysis ability (standard curve correlation between antigen concentration and signal intensity>0.95 ), providing a reliable technical platform for parallelism and multi-factor protein quantitative detection. In addition, the chip is stable after spotting, and the activity can be maintained for at least 2 months, which provides important conditions for the industrialization and scale of the chip.

Description

1. Technical field [0001] The invention belongs to the technical field of protein chip manufacturing, in particular to a method for constructing an antibody microarray based on double-antibody sandwich immunoassay. 2. Background technology [0002] Proteomics is the study of the quantity, structure, and cellular function of all proteins in an organism, organ, and organelle, as well as their changes in different time, space, and physiological states. Currently, proteomics techniques are mainly divided into unbiased proteomics and biased proteomics. Among them, unbiased proteomics is a "discovery-oriented" science. Analyzing all proteins in a sample can obtain a large amount of data for discovering unknowns, mainly two-dimensional gel electrophoresis combined with mass spectrometry. . Biased proteomics, also known as system-oriented proteomics, is based on the researchers' understanding of a specific pathophysiological process, presupposing that certain components in the sam...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/543G01N21/64
Inventor 刘必成吕林莉陆祖宏
Owner SOUTHEAST UNIV
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