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Construction method of antibody microarray based on double-antibody sandwich immunanalysis

A double-antibody sandwich and immunoassay technology, used in analytical materials, material excitation analysis, material testing products, etc.

Inactive Publication Date: 2007-07-18
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 immunanalysis
  • Construction method of antibody microarray based on double-antibody sandwich immunanalysis
  • Construction method of antibody microarray based on double-antibody sandwich immunanalysis

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 30 minutes, washed with 0.1M pH7.4 PBS for 5 minutes*3 times, blown dry by nitrogen flow, and applied immediately; 3. Spotting and fixing of capture antibody (step 1 of attached picture 1): preparation of spotting buffer: 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 (20...

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 4Spotting 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 setting-up antibody micro-array based on double-antibody sandwich immunological analysis includes preparing carrier, activating carrier, fixing point sample of catching antibody and immunological analysis based on micro-array.

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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IPC IPC(8): G01N33/68G01N33/543G01N21/64
Inventor 刘必成
Owner SOUTHEAST UNIV
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