Signal amplification immunodetection method

A technology for signal amplification and detection, which is applied in the biological field and can solve problems such as being unable to be realized at the same time.

Inactive Publication Date: 2016-03-02
CHANGZHOU BIOWIN BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] High sensitivity, rapidity, miniaturization, full quantification, and automation are the current development trends of clinical immunoassay technology products, but none of the existing ones can achieve the above functions at the same time

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1, the preparation of filter

[0070] 1. Experimental materials

[0071] Long columnar microfilter housing (height 25 mm, diameter 5 mm), NHS-activated agarose gel particles (ACROBIOSYSTEMS), anti-human fibrinogen polyclonal antibody (Genagates, whose catalog number is GP1032), sodium bicarbonate, hydrochloric acid , Ethanolamine.

[0072] 2. Experimental method

[0073] Take 5mg of anti-human fibrinogen polyclonal antibody and add 2ml of 0.2M aqueous sodium bicarbonate solution (pH8.3) to dissolve it. Take 1ml of NHS-activated agarose gel particles, wash with 20ml of 1mM hydrochloric acid, and filter three times to remove all the hydrochloric acid solution. The anti-human fibrinogen polyclonal antibody solution was mixed with the treated NHS-activated agarose gel particles at a ratio of 1:1 by volume, and reacted with shaking at 4°C for 4 hours. Wash the reacted NHS-activated agarose gel particles with pure water, then add a solution containing 10mM etha...

Embodiment 2

[0074] Embodiment 2, the comparison between the detection performance of the present invention and existing chemiluminescence detection technology:

[0075] 1. Experimental materials

[0076] Anti-human fibrinogen polyclonal antibody filter prepared in Example 1, horseradish peroxidase-labeled anti-human fibrinogen monoclonal antibody (Genagates company, its catalog number is GL1101), magnetic particles (MP-COOH-20020 , Zhengzhou Inno Biotechnology Co., Ltd.), luminol, p-iodophenol, carbamide peroxide, chemiluminescence detector (Promega, GlomaxMultiJRDetectionSystem), human fibrinogen (Sigma-Aldrich product, catalog number is F3879-1G) .

[0077] 2. Experimental method

[0078] Preparation of human fibrinogen solution: Take human fibrinogen solution of known concentration and dilute it with PBS solution to prepare 1 μg / ml human fibrinogen solution.

[0079] Labeling of magnetic particles: using a conventional labeling method, label the magnetic particles with 1 mg / ml anti-...

Embodiment 3

[0091] Embodiment 3, the comparison between the present invention and the detection result of existing chemiluminescence detection technology

[0092] 1. Experimental materials

[0093] Anti-human fibrinogen polyclonal antibody filter prepared in Example 1, horseradish peroxidase-labeled anti-human fibrinogen monoclonal antibody (Genagates company, its catalog number is GL1101), anti-human fibrinogen polyclonal antibody (Genagates company, its product catalog number is GP1032), magnetic particles (MP-COOH-20020, Zhengzhou Inno Biotechnology Co., Ltd.), luminol, p-iodophenol, carbamide peroxide, chemiluminescence detector (Promega, GlomaxMultiJRDetectionSystem ), human fibrinogen (product of Sigma-Aldrich, catalog number F3879-1G), healthy human plasma (donated by healthy volunteers).

[0094] 2. Experimental method

[0095] Preparation of human fibrinogen solution: Take human fibrinogen solution of known concentration and dilute it with PBS solution to prepare a series of hu...

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Abstract

The invention discloses a signal amplification immunodetection method. According to the method, signal amplification immunity quantitative detection is conducted through a filter, and the method comprises the step of adopting the filter for sequentially filtering a to-be-detected sample solution A containing objects to be detected, a detected object solution B labeled through avidin and an indicator solution C labeled through biotin, wherein a filter element of a filter layer of the filter is a solid phase material coupled to a specific binder of the objects to be detected, detected objects labeled through the avidin can be specifically bound to the objects to be detected, and thereby the quantity of the objects to be detected in the solution A is calculated according to the color or light quantity variation of an indicator. It is proved through experiments that compared with a conventional biotin-avidin amplification reaction sequence, the avidin-biotin amplification reaction sequence has a greater biological signal amplification effect. The method can be used for detecting samples which are from human bodies and animal bodies and can be subjected to disease diagnosis and health detection and applied to development of products for sample quantitative immunodetection in the fields of environment, pharmaceutical analysis, food and industrial analysis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a signal amplification immunoassay method. Background technique [0002] Immunological detection technology is an experimental method designed by applying the principles of immunology to determine antigens, antibodies, immune cells and chemical components. Samples for analytical, food and industrial analysis. Commonly used immunoturbidity technology, solid-phase enzyme immunoassay technology, chemiluminescent detection technology, immunofluorescence labeling technology, flow cytometry, colloidal gold technology, etc. [0003] Immunoturbidimetry, also known as immunoturbidimetry, is the specific combination of soluble antigens and antibodies in the liquid phase to produce complexes of a certain size, which form light refraction or absorption, and measure the transmitted or scattered light after refraction or absorption. As a calculation unit, it is used for quantitative detection, but...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/574G01N33/68
Inventor 刘凤鸣李亚星李金萍曹勤燕
Owner CHANGZHOU BIOWIN BIOPHARM
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