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Captured-antibody competition sandwich immunodetection method capable of extending detection scope and biosensor

A biosensor and capture antibody technology, applied in the field of immunodetection, can solve the problems of inability to achieve detection sensitivity, inability to simplify operation steps, and high detection upper limit, and achieve the effects of short detection time, simple operation and small sample volume

Inactive Publication Date: 2015-05-06
TECHNICAL INST OF PHYSICS & CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The amount of labeled antigen bound to the immobilized capture antibody is negatively correlated with the concentration of the antigen to be tested in the sample. This method has a high detection limit, but cannot achieve the sensitivity of the detection limit and cannot simplify the operation steps.

Method used

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  • Captured-antibody competition sandwich immunodetection method capable of extending detection scope and biosensor
  • Captured-antibody competition sandwich immunodetection method capable of extending detection scope and biosensor
  • Captured-antibody competition sandwich immunodetection method capable of extending detection scope and biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of magnetic particle CRP biosensor test card

[0047] 1. Preparation of magnetic particle-labeled antibody: first use NHS-Biotin to label CRP antibody, NHS-Biotin is coupled with antibody through NHS to form Biotin-CRP antibody, and then use Avidin-magnetic particles and Biotin-CRP antibody to pass Avidin and Biotin Coupled with the interaction of magnetic particles to obtain CRP antibody labeled with magnetic particles. The preparation steps of magnetic particle-labeled CRP antibody are as follows:

[0048] (1) Prepare 10 mM NHS-Biotin in DMF.

[0049] (2) Ultrafiltration removes sodium azide in the CRP antibody.

[0050] (3) Mix the CRP antibody and NHS-Biotin at a molar ratio of 1:50, incubate and shake at 4°C for 2 hours (or keep it overnight) to couple the CRP antibody to Biotin to obtain Biotin-CRP antibody.

[0051] (4) The above mixed solution was ultrafiltered at 4° C. under the condition of 9000 rpm to remove unreacted NHS-Biotin.

...

Embodiment 2

[0057] Example 2: Preparation of magnetic particle CRP biosensor test card

[0058] A capture antibody competition sandwich immunoassay biosensor with extended detection range, including a sensor test card 100, the sensor test card 100 includes a cover plate 130, a support plate 170 and a bottom plate 140, and the support plate 170 is located between the cover plate 130 and the bottom plate 140 , the support plate 170 is provided with at least one microchannel 150, the microchannel includes a reagent storage area 160, an immune reaction area 120 and a waste liquid area 180; the immune reaction area 120 contains immobilized capture antibodies, and the reagent storage area 160 A liquid injection hole 110 is provided on the cover plate corresponding to the position of the waste liquid area 180 , and a liquid discharge hole 190 is provided on the cover plate corresponding to the position of the waste liquid area 180 . Wherein the reagent storage area 160 contains free capture anti...

Embodiment 3

[0059] Example 3: Preparation of magnetic particle CRP biosensor test card

[0060] The capture antibody competition sandwich immunoassay biosensor with expandable detection range includes a sensor test card 200, the sensor test card 200 includes a cover plate 230 and a bottom plate 240, a microchannel 250 is formed between the cover plate 230 and the bottom plate 240, and the microchannel 250 is formed between the cover plate 230 and the bottom plate 240 The channel 250 includes an immunoreaction zone 220 containing immobilized capture antibodies and a sensor zone 260 , and the cover plate has an injection / drain hole 210 and a drain / injection hole 210 at each end.

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Abstract

The invention discloses a captured-antibody competition sandwich immunodetection method capable of extending detection scope and a biosensor. The captured-antibody competition sandwich immunodetection method is utilized for detecting the concentration of a to-be detected antigen, concretely, the antigen in a to-be detected sample is firstly reacted with a dissociated captured antibody and a marked antibody, and then reacted with an immobilized captured antibody, so that only one complex of marked antibody-antigen-immobilized captured antibody in formed immunization complexes can be detected, and the antigen concentration is determined by measuring the mark signal on the marked antibody of the immunization complex. Because the dissociated captured antibody-antigen-marked antibody complex and the dissociated captured antibody-antigen complex cannot be detected by a detector, the purpose of indirectly diluting the antigen concentration in the to-be detected sample is realized, and the detection scope of the antigen concentration of the to-be detected sample is expanded. Also, the biosensor prepared by utilizing the method has the advantages of wide detection scope, high sensitivity and short detection time.

Description

technical field [0001] The invention relates to the technical field of immunoassay, more specifically, to a capture antibody competition sandwich immunoassay method and a biosensor with an expandable detection range. Background technique [0002] Immunological detection is the use of immunological principles to detect the substances to be tested in samples, and its methods can be divided into qualitative methods and quantitative methods. Immunoassay has the advantages of specificity, simplicity, speed, accuracy and sensitivity. According to different detection principles, immunoassay can be divided into indirect method, sandwich method (including double antigen sandwich method and double antibody sandwich method), capture method, competition method, etc. , each method has its own advantages and limitations. [0003] The principle of the traditional double-antibody sandwich immune reaction (sandwich immunoassay) is to use the antigen as the detection target. The surface of ...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/531
CPCG01N33/53G01N33/54306
Inventor 高云华高宇哲
Owner TECHNICAL INST OF PHYSICS & CHEMISTRY - CHINESE ACAD OF SCI
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