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Method for quantitative determination of specific analyte with single trapping agent and reagent kit therefor

An analyte and capture agent technology, applied in the field of bioengineering, can solve the problems of cumbersome detection methods, difficult protein labeling, inhibition of antibody binding, etc., and achieve the effects of high detection specificity, wide application range, and low noise

Inactive Publication Date: 2005-11-23
孙东旭
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

If hundreds of thousands or even tens of thousands of proteins are quantitatively detected at the same time, it is necessary to label the detection antibody of each protein, which is a huge workload, and it will cause different batches of detection due to different manual labeling conditions and efficiencies. Difference Between Antibodies
In addition, chemical modifications of the labeling process may affect the binding of the detection antibody to the antigen
Third, when detecting multiple proteins in multiplex, it is necessary to mix the detection antibodies corresponding to all proteins together, resulting in the dilution of each single antibody and the increase of non-specific binding, reducing the specific signal and sensitivity, and expanding detect noise
But the disadvantages are, first, biological samples contain tens of thousands of large and small molecules, many of which will interfere with the labeling efficiency, and proteins with low content are often difficult to be effectively labeled
Second, the labeling process itself may modify and change the characteristics of the epitope on the protein molecule, reducing or even inhibiting the binding to the antibody
Experimental results have proved that antigen pre-labeled detection methods usually have a large background noise and low sensitivity
In order to improve the detection sensitivity, it has recently been proposed to label the detection antibody with DNA oligonucleotides in the Sandwich ELISA method, and then use polymerase chain reaction (PCR) or rolling circle replication (rolling circle replication) method to amplify the signal, these Although the method can improve the detection sensitivity to a large extent, the detection method is too cumbersome and the cost is too high, and several limitations of the above-mentioned Sandwich ELISA method still exist

Method used

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  • Method for quantitative determination of specific analyte with single trapping agent and reagent kit therefor
  • Method for quantitative determination of specific analyte with single trapping agent and reagent kit therefor
  • Method for quantitative determination of specific analyte with single trapping agent and reagent kit therefor

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Embodiment 1

[0035] Example 1: Singleness detection (detection of a protein)

[0036] In this embodiment, both the capture device and the detection device are 96-well microtiter plates. Each well of the capture device is coated with an antibody that detects an antigenic protein. Antigen proteins to be tested are four cytokines, namely IL-1-beta, IL-4, IL-8 and GM-CSF. The corresponding antibodies are all monoclonal antibodies.

[0037] (1) Capture antigenic protein:

[0038] a. Coated capture antibody: Take two 96-well microplates (flat bottom, high binding to protein), one for capturing antigen in the sample (capture device), and the other for detecting labeled antigen (detection device). 100 μl of monoclonal antibodies against IL-1-beta, IL-4, IL-8 or GM-CSF at a concentration of 0.5 μg / ml (diluted in PBS buffer) were added to the microwells. Only one antibody was added to each microwell, and each antibody was added to 8 microwells on each microplate. Place the microplate at 4 overn...

Embodiment 2

[0050] Embodiment 2: Multiplex detection (simultaneous detection of three proteins)

[0051] In this embodiment, both the capture device and the detection device are 96-well microtiter plates. The surface of each microwell of the capture device is coated with three kinds of antibodies mixed together to detect three antigenic proteins simultaneously. Antigen proteins to be tested were three cytokines, namely IL-1-beta, TNF-α and IL-10. The corresponding antibodies are all monoclonal antibodies.

[0052] (1) Capture antigenic protein:

[0053] a. Coating capture antibody: Take two 96-well microplates (flat bottom, high protein binding), one for capture (capture device) and one for detection (detection device). Add 100 μl of three antibody mixtures against IL-1-beta, TNF-α and IL-10 into the microwells of the capture device, the concentration of each antibody is 0.5 μg / ml (diluted in PBS buffer), add a total of 8 micropores. However, only one antibody is added to each microw...

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Abstract

The invention relates to a method and its medium box for quantity measuring peculiar analysis substance by single capture medium. The method comprises the following steps: combining the tested analysis substance with solid phase capture medium, labeling analysis substance which has been captured by capture medium with report molecule, cleaning-up analysis substance from compound, recombining tested analysis substance with new solid phase capture medium, ascertaining content of analysis substance by report molecule's label signal. The medium box comprises capture device, testing device report molecule used as label and analysis substance eluent.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for quantitatively detecting specific analytes with a single capturing agent and a kit thereof. Background technique [0002] Detecting the content of specific protein factors in biological (including human) tissue samples is of great significance for applications and basic research in the fields of medicine, biology, agriculture, and the like. For example, the detection of specific protein components of pathogenic microorganisms is currently an important means of diagnosing infectious diseases; similarly, the determination of changes in the levels of early biomarker protein factors in cancer is extremely important for early detection and treatment of diseases and observation of curative effects. According to different application goals, the analyte to be tested can be one or several proteins, or a group of proteins with different numbers. In recent ye...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/577
CPCG01N33/54306
Inventor 孙东旭
Owner 孙东旭
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