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Immunofluorescence label reagent kit

A technology of immunofluorescence and labeling reagents, which is applied in the field of immunoassay and biotechnology applications, can solve the problems of photobleaching, non-specific staining, and affecting the sensitivity of immunoassays, and achieve the effect of good sensitivity, reliability and easy operation

Inactive Publication Date: 2008-03-26
SHANDONG UNIV AT WEIHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has its disadvantages: a large number of antibodies or antigen molecules and enzymes participate in coupling as reactants; purification steps are required to obtain antibody-enzyme conjugates; enzyme activity often decreases after cross-linking; Interference caused by steric hindrance affects the sensitivity of immunoassays
In addition, traditional fluorescent dyes are prone to photobleaching under the excitation light of a fluorescent microscope, and traditional antibody preparation and labeling methods tend to reduce antibody titers and cause non-specific staining

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Immunofluorescence labeling kit

[0027] The composition is as follows: fusion protein SPA-EGFP composed of enhanced green fluorescent protein and staphylococcal protein A, blocking agent 0.5% bovine serum albumin, elution buffer containing 10mMPBS 0.5% Tween 20, pH7.4.

Embodiment 2

[0028] Example 2: Preparation of Immunofluorescence Labeling Kit

[0029] 1. For the method of constructing and expressing the fusion protein SPA-EGFP, refer to CN1731181.

[0030] The SPA-EGFP fusion protein gene spa-egfp (plasmid is a DNA sequence, only the gene can be inserted into the plasmid) is inserted downstream of the α-factor signal peptide of the plasmid pPIC9K, the host cell for expression is Pichia pastoris GS115, and Pichia pastoris is electrotransformed and Screen high-copy recombinants and construct recombinant SPA-EGFP fusion gene Pichia pastoris engineering bacteria. The above-mentioned Pichia pastoris engineering bacteria were fermented, using BMMY medium, and the fermentation conditions were: 30° C., methanol concentration 2%, buffer solution pH value 7.0, and 1% Casaminoacids were added at the same time. Cultivate on a shaker at 300 rpm for 80-84 hours, and add methanol every 20-24 hours to a final concentration of 2%. Centrifuge the fermentation broth o...

Embodiment 3

[0035] Example 3: The method of using the immunofluorescence labeling kit (direct method)

[0036]Dilution: Dilute the antigen to a certain concentration with dilution buffer as needed.

[0037] Fixation: Drop the diluted antigen onto the solid phase carrier, cover the antigen-free area with a blocking agent, and avoid non-specific adsorption of the fluorescent indicator.

[0038] Labeling: Dilute the fluorescent indicator to the concentration required for the experiment, mix it with the sample, and incubate it to allow it to bind to the antibody.

[0039] Sample loading: drop the labeled sample onto the solid phase carrier immobilized with the antigen, and incubate to allow the antigen and antibody to bind.

[0040] Washing: wash the solid phase carrier with elution buffer three times to remove unbound samples and fluorescent indicators.

[0041] Observation of results: Qualitative / quantitative observation under a fluorescent microscope.

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PUM

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Abstract

This invention discloses to a kind of immunofluorescence mark reagent box. It is a reagent box applied for the aspects of immunity mark which uses the green fluorescence protein (GFP) as the indicator, the staphylococcus protein A (SPA) as the antibody. It also discloses the preparation method of the reagent. This invention can applied wide range in the field such as the immunity fluorescence mark, immunity histochemistry, biochemistry analysis, antibody test, protein chip and so on. It can replace the exist product efficiently, its operation is more simple, stability, delicacy and reliability, it is especially the same with the high flux analysis which analyze by the fluorescence analysis instrument.

Description

technical field [0001] The present invention uses green fluorescent protein as an indicator, staphylococcal protein A as an anti-antibody, and is applied to a kit for immunolabeling, which relates to immunofluorescent labeling, immunohistochemistry, biochemical analysis, antibody detection, protein fusion, and green fluorescence Protein, protein A, genetic engineering, etc., belong to the related fields of immunoassay and biotechnology application. Background technique [0002] Since the emergence of labeled immunoassay technology, it has been widely used in medical and biological research. The principle is to combine the specificity and sensitivity of the antigen-antibody reaction with the accuracy of microscopic tracing, label the antibody with chromogenic or fluorescent substances, and detect the antigenic substances in tissues or cells. [0003] In immunoassays, it is necessary to label the antibody / antigen with an enzyme or fluorescent substance. The traditional label...

Claims

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Application Information

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IPC IPC(8): G01N33/52G01N33/53G01N33/533
Inventor 吉爱国梁浩宋超
Owner SHANDONG UNIV AT WEIHAI
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