Multiple signal amplification system and application thereof in immunospot detection

A technique of amplifying system and multiple signals, applied in the field of immunodetection, which can solve problems such as low sensitivity

Active Publication Date: 2021-05-18
艾克发(北京)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although FluoroSpot can detect two antigens at one time, it can only perform one round of detection and the sensitivity is low due to the lack of amplification system

Method used

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  • Multiple signal amplification system and application thereof in immunospot detection
  • Multiple signal amplification system and application thereof in immunospot detection
  • Multiple signal amplification system and application thereof in immunospot detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0355] Example 1 Using one-level multiple amplification system and immunospot method to detect antigen

[0356] In this example, the one-level multiple amplification system was applied to the immunospot method to simultaneously detect six kinds of antigens secreted by activated cells, and the six kinds of antigens were IL-10, TGF-β, IFN-γ, and TNF-α , IL-2 and IL-6.

[0357] 1. Preparation of antibody and DNA linker (take the connection of Anti-human IL-10antibody and DNA linker 1 as an example)

[0358] (1) Concentrate the antibody (Anti-human IL-10antibody) to 2mg / mL through an ultrafiltration centrifuge tube (100kDa MWCO) to remove azide and other preservatives.

[0359] (2) The antibody was exchanged into PBS buffer (pH7.4) through a desalting spin column (7000Da MWCO).

[0360] (3) Antibody and LC-SMCC (succinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxy-[6-aminocaproate]) (molar ratio 1 :20) reacted at 4°C for 3 hours.

[0361] (4) Remove excess connection inter...

Embodiment 2

[0388] Example 2 Using two-stage multiple amplification system and immunospot method to detect antigen

[0389] In this example, the two-stage multiple amplification system was applied to the immunospot method to simultaneously detect six antigens secreted by cells, the six antigens were IL-10, TGF-β, IFN-γ, TNF-α, IL- 2 and IL-6.

[0390] 1. Preparation of antibody and DNA linker

[0391] In this example, the amino group on the antibody protein is reacted with GMBS (N-[γ-maleimide butyryloxy]succinimide ester), and then GMBS is used to connect the sulfhydryl group on the DNA linking chain. The antibody is linked to the DNA linker, and the linking intermediate used has a bifunctional group, one end can be linked to the antibody, and the other end can be linked to the DNA. The specific experimental steps are the same as in Example 1.

[0392] 2. Preparation of the DNA primary amplification chain in the signal amplification system

[0393] The preparation process and specifi...

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Abstract

The invention relates to a signal amplification system and application thereof in immunospot method detection. The invention aims to provide a method for performing immunospot method detection by using the multiple signal amplification system. The method comprises the following steps of: (1) fixedly a coated antibody or antigen on a solid-phase substrate; (2) adding to-be-detected cells and a cell stimulant or directly adding the to-be-detected cells which have acted with the stimulant; (3) adding an antibody-DNA / RNA connecting chain or an antigen-DNA / RNA connecting chain, and acting for a certain time; (4) adding a DNA / RNA amplification chain, and acting for a certain time; (5) adding a DNA / RNA detection chain, and reacting for a certain time; (6) performing washing, and detecting the intensity of to-be-detected signals on the DNA / RNA detection chain; and (7) quantitatively analyzing the number of immune spots with detection signals formed on the solid-phase substrate. The detection method disclosed by the invention has the advantages of multiple amplification of signals, low detection limit, higher sensitivity, capability of simultaneously detecting a plurality of target objects, multi-round detection, simplicity and convenience in operation and the like.

Description

technical field [0001] The invention belongs to the field of immune detection, and in particular relates to a multiple signal amplification system based on DNA / RNA complementary pairing and its use in the detection of immunospot method. Background technique [0002] Immunospot method refers to an immunological detection method that binds antibodies to solid phase supports such as PVDF and aldehyde-formed porous plates, and then uses antigen-antibody specific binding to detect cytokines and other substances secreted by cells. Enzyme-linked immunospot method (ELISPOT) is the most commonly used immunospot method. Through the color reaction, clear and identifiable spots appear on the corresponding positions of the cells to be detected that secrete soluble proteins and other substances. The spots can be directly counted manually under the instrument or The spots are counted by the ELISPOT analysis system, and one spot represents one active cell, so as to calculate the number of c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543C12Q1/682C12Q1/6804C12N15/11
CPCG01N2458/10G01N33/6854G01N33/54306G01N33/532Y02A50/30
Inventor 刘密彭作富
Owner 艾克发(北京)生物技术有限公司
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