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Method for improving signal-to-noise ratio of antibody and fluorescent protein directional coupling marker

A fluorescent protein and signal-to-noise ratio technology, applied in the field of immunology, can solve the problems of strong background signal, low signal-to-noise ratio, and less phycoerythrin, and achieve the effect of reducing negative signal and improving signal-to-noise ratio

Pending Publication Date: 2021-04-09
ZHEJIANG ZHENGXI BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Since the target protein and phycoerythrin carry both amino groups and sulfhydryl groups, when the traditional amine-sulfhydryl cross-linking agent such as SMCC is used for activation, the amine-sulfhydryl cross-linking agent will not only bind to amino groups, but also bind to sulfhydryl groups. This causes that when the protein activated by the amine-sulfhydryl crosslinker is cross-linked with the thiolated protein, it is difficult for the crosslinker on the protein activated by the amine-sulfhydryl crosslinker to bind to the sulfhydryl group to recrosslink the thiol protein. Linking, resulting in less phycoerythrin bound to the target protein, poor labeling effect, weak positive signal and strong background signal of the obtained immunofluorescence probe, and low signal-to-noise ratio during detection

Method used

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  • Method for improving signal-to-noise ratio of antibody and fluorescent protein directional coupling marker
  • Method for improving signal-to-noise ratio of antibody and fluorescent protein directional coupling marker
  • Method for improving signal-to-noise ratio of antibody and fluorescent protein directional coupling marker

Examples

Experimental program
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Effect test

Embodiment 1

[0032]In this example, a method of preparing an alginate immunofluorescence probe prepared by anti-mouse CD4 monoclonal antibody [GK1.5] (hereinafter referred to as CD4 antibody), the preparation method includes the following steps:

[0033](1) The amino group on the target protein is reacted with Sulfo-S-Hynic;

[0034]Specifically include:

[0035]0.7 ul 60mm sulfo-s-hynic solution was added to 200 ug CD4 antibody, and after mixing, it was placed at room temperature at room temperature for 1.5 h;

[0036]In this step, the Sulfo-S-Hynic molecule binds to the free amino group in the CD4 antibody molecule to prepare Sulfo-S-Hynic-CD4;

[0037]The reaction liquid obtained by step (1.1) was shifted into the ultrafiltration centrifuge tube, and then 500 ul of sodium phosphate (pH 6.0) buffer, 12000 g centrifuge for 5 min and remove the filtrate, and then 500 ul of sodium phosphate (pH 6.0) buffer buffer mixed Centrifuge, repeat this operation 5 times and the antibody is disposed to 46 ul;

[0038]This st...

Embodiment 2

[0051]In this example, an alginate immunofluorescence probe preparation method (closed on the basis of Example 1) was introduced as a target protein in anti-mouse CD4 monoclonal antibody [GK1.5] (hereinafter referred to as CD4 antibody). This preparation method includes the following steps:

[0052](1) The amino group on the target protein is reacted with Sulfo-S-Hynic;

[0053]Specifically include:

[0054](1.1) Add 0.7 ul 60mm sulfo-s-hynic solution to 200 ug CD4 antibody, mixed up, at 25 ° C, at room temperature, 1.5 h;

[0055]In this step, the Sulfo-S-Hynic molecule binds to the free amino group in the CD4 antibody molecule to prepare Sulfo-S-Hynic-CD4;

[0056](1.2) Removal of the reaction liquid obtained by step (1.1) to the ultrafiltration centrifuge tube, then add 500 ul of sodium phosphate (pH.0) buffer, 12000 g from 5 min and remove the filtrate, and then add 500 ul of sodium phosphate (pH 6.0) buffer. Mix centrifugation and repeat this operation 5 times and the antibody is disposed to ...

Embodiment 3

[0070]In this example, an alginate immunofluorescence probe preparation method (closed on the basis of Example 1) was introduced as a target protein in anti-mouse CD4 monoclonal antibody [GK1.5] (hereinafter referred to as CD4 antibody). This preparation method includes the following steps:

[0071](1) The amino group on the target protein is reacted with Sulfo-S-Hynic;

[0072]Specifically include:

[0073](1.1) Add 0.7 ul 60mm sulfo-s-hynic solution to 200 ug CD4 antibody, mixed up, at 25 ° C, at room temperature, 1.5 h;

[0074]In this step, the Sulfo-S-Hynic molecule binds to the free amino group in the CD4 antibody molecule to prepare Sulfo-S-Hynic-CD4;

[0075](1.2) Removal of the reaction liquid obtained by step (1.1) to the ultrafiltration centrifuge tube, then add 500 ul of sodium phosphate (pH.0) buffer, 12000 g from 5 min and remove the filtrate, and then add 500 ul of sodium phosphate (pH 6.0) buffer. The buffer is mixed with centrifugation and repeats this operation 5 times and the an...

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Abstract

The invention relates to a method for improving the signal-to-noise ratio of an antibody and fluorescent protein directional coupling marker. The method comprises the following steps of: step 1) making PE react with a first cross-linking agent; 2) making the antibody react with a second cross-linking agent; and (3) performing cross-linking reaction on the cross-linked PE in the step (1) and the cross-linked antibody in the step (2). The first cross-linking agent is Sulfo-S-HyNic or S-HyNic, the second cross-linking agent is Sulfo-S-4FB or S-4FB, or when the first cross-linking agent is Sulfo-S-4FB or S-4FB, the second cross-linking agent is Sulfo-S-HyNic or S-HyNic. When being applied to immunodetection, the method is strong in signal strength and high in signal to noise ratio.

Description

Technical field[0001]The present invention belongs to the field of immunology, and more particularly to a method of immunofluorescent protein labeling such as alginate.Background technique[0002]Algae (P-PHYCOERYTHRIN) is a novel fluorescent labeling reagent that is prevalently used from the purification of purification from red algae. Under the excitation of a particular wavelength, the algae can emit strong fluorescence, and the fluorescence intensity is 30-100 times the fluorescein, with good absorbing performance and high quantum yield, which has a wide excitation in the visible spectral area. And launch range.[0003]The algae red protein is used in fluorescence analysis, and has the advantages of conventional chemical fluorescent dyes that cannot be compatible. For example, (1) has a wide absorption spectrum in a wider pH range, more likely to select a suitable excitation wavelength, thereby obtaining high efficiency fluorescence emission, and there is a specific fluorescence emi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/577G01N33/58G01N33/68G01N33/569
CPCG01N33/533G01N33/577G01N33/582G01N33/68G01N33/56966G01N2333/70514
Inventor 张洋
Owner ZHEJIANG ZHENGXI BIOMEDICAL CO LTD