ELISA (Enzyme-Linked Immunosorbent Assay) detection method of activation level of complement system

A complement system and detection method technology, applied in the field of immunological detection, can solve the problems of slow research speed, heavy workload, and expensive reagents

Inactive Publication Date: 2013-06-26
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method can be used to determine the site of action of drugs in the complement activation system, but most of the complement-deficient serum needs to be prepared by itself, the steps are cumbersome and complicated, and the workload is heavy, and the research speed is slow
If the complement deficient serum is purchased directly, the reagents are expensive
At the same time, this method cannot study the non-specific effect of drugs on the complement system or the semi-hemolytic effect, which has certain defects.

Method used

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  • ELISA (Enzyme-Linked Immunosorbent Assay) detection method of activation level of complement system
  • ELISA (Enzyme-Linked Immunosorbent Assay) detection method of activation level of complement system
  • ELISA (Enzyme-Linked Immunosorbent Assay) detection method of activation level of complement system

Examples

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Embodiment 1

[0038] Example 1 Determination of the effect of suramin on the activation level of classical pathway and mannose pathway complement

[0039] The ELISA plate in this example is a 96-well single-well detachable ELISA plate purchased from Nunc Company, and the coating buffer is 0.1mol / L carbonate buffer at pH 9.6, classical pathway and mannose pathway The guinea pig serum and the test drug buffer are BVB at pH 7.5 ++ Buffer, Alternative Route Guinea Pig Serum and Drug to be Tested The buffer is GVB / Mg++-EGTA buffer at pH 7.5, and the washing solution is 0.05% PBS-T buffer at pH 7.4. Commercial rabbit anti-human C3c polyclonal antibody and goat anti-rabbit IgG-HRP enzyme-labeled antibody were purchased from Shanghai Changdao Biotechnology Co., Ltd. Suramin (Sigma)

[0040] 1. Method:

[0041]Coat the ELISA plate with 5 μg / ml human IgM and 100 μg / ml mannose coating solution respectively, 50 μl / well, overnight at 4°C; wash 3 times with PBS-T, 250 μl / well, shake gently and pat on ...

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Abstract

The invention belongs to the field of an immunological detection method and discloses an ELISA (Enzyme-Linked Immunosorbent Assay) detection method of an activation level of a complement system. Aiming at classic, replaced and mannose activation ways of the complement system, three different activators are respectively used for covering a 96-pore high-adsorbability elisa plate to activate the complement system in vitro; a polyclonal anti-complement antibody is used as an intermediate for capturing an activated complement; and anti-IgG-HRP (Horseradish Peroxidase) is used as an ELISA detection antibody so as to reflect an activation condition of the complement system. According to the ELISA detection method disclosed by the invention, a two-step method is used as an amplification system to replace a traditional three-step method. The method disclosed by the invention consumes less time and is simple in operation process; and a step of marking the antibody by digoxin can be saved so that an experiment is more convenient. The method disclosed by the invention makes up the detects that an existing hemolytic test cannot determine the activation conditions of a complement mannose way, and is particularly suitable for detecting the inhibition effect on the complement system by medicines; and a brand-new rapid and efficient complement inhibition agent in-vitro screening method is established.

Description

technical field [0001] The invention belongs to the field of immunological detection methods, and in particular relates to an ELISA detection method for drugs affecting the activation levels of three activation pathways of the complement system. Background technique [0002] The prior art discloses that the complement system includes more than 30 components, which are widely distributed in the serum, interstitial fluid and cell membrane surface of humans and vertebrates, and is a protein reaction system with a precise regulation mechanism. Complement is not only an important part of the body's innate immune defense, but also one of the main mechanisms for antibodies to exert immune effects. Studies have shown that the normal activation of the complement system can mediate defense against microbial infection and inflammatory responses, but its abnormal activation can lead to serious damage to the body. Observing the dynamic changes of complement clinically is of great signif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
Inventor 力弘章蕴毅吴牧鹭陈道峰
Owner FUDAN UNIV
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