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Dendritic structure mark and preparation method thereof

A dendritic structure and marker technology, which is applied in biochemical equipment and methods, microbial determination/inspection, fluorescence/phosphorescence, etc., to reduce non-specific reactions, simplify preparation methods, and enhance sensitivity

Inactive Publication Date: 2008-09-03
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Dendritic structure mark and preparation method thereof
  • Dendritic structure mark and preparation method thereof
  • Dendritic structure mark and preparation method thereof

Examples

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preparation example Construction

[0039] 1. Preparation of biotin-labeled double-stranded oligonucleotide

[0040] Use 2×SSC (0.3M sodium citrate, 30mM sodium chloride, pH7.0) buffer solution to mix biotin-labeled single-stranded deoxyribonucleic acid (Invitrogen, Carlsbad, CA) and its complementary nucleic acid chain (Invitrogen, Carlsbad, CA), for example, the two chains are: chain A: biotin-5'-TTT TTT TTT GCG GGT AAC GTC AAT ATT AAC TTT ACT CCC-3', chain B: 5'-GGG AGT AAA GTT AAT ATT GAC GTT ACC CGC-3'. Denature at 95°C for 5 minutes, cool to room temperature naturally, and determine the concentration of nucleic acid after hybridization with a spectrophotometer.

[0041] 2. Fluorescein isothiocyanate (FITC) labeled antibody or protein molecule

[0042] Mouse immunoglobulin G (mIgG), goat anti-mouse immunoglobulin G and streptavidin (SA) dissolved in carbonate buffer (50mM sodium carbonate, 50mM sodium bicarbonate, pH 9.15), respectively Mix with FITC (5 mg FITC / mL DMSO) dissolved in dimethyl sulfoxide (DMSO) at...

Embodiment 1

[0045] Example 1. Optimization of coating conditions for mouse immunoglobulin G

[0046] Take 100 μl of FITC-labeled mouse immunoglobulin G (FITC-mIgG) with concentrations of 0, 0.3, 1, 3, 10, 30, 100 μg / mL, respectively, and dissolve it in a buffer containing 50 mM sodium bicarbonate and a pH of 9.6 In the medium, a 96-well plate was coated according to the amount of 100μl per well at 4°C overnight. After washing 3 times with a buffer containing 50mM 3-hydroxymethyl-aminomethane, 50mM sodium chloride and 0.1% Tween 20, pH 8.0, the optimal fluorescence detection conditions of FITC (i.e. the optimal fluorescence excitation wavelength , Excitation slit width, fluorescence emission wavelength, emission slit width), detect the change of fluorescence intensity to indicate the saturation concentration of FITC-mIgG, and use this saturation concentration as the coating concentration of unlabeled mIgG.

Embodiment 2

[0047] Example 2. Optimization of experimental conditions for biotin-labeled goat anti-mouse immunoglobulin G

[0048] Coat mIgG with optimized mIgG coating conditions at 4°C overnight. After washing 3 times, add 300 μl of 1% bovine serum albumin-containing phosphate buffer at 4°C overnight. After washing 3 times, add 100μl of FITC-labeled goat anti-mouse IgG with concentrations of 0, 1.0, 3.2, 9.7, 29.0, 87.1, 261.3, 784.0, 2352.0μg / mL, 37℃, air bath constant temperature 200rpm shaking for 2 hours; washing After 3 times, the optimal fluorescence detection conditions of FITC were used to detect the change of fluorescence intensity to indicate the saturation concentration of FITC-mIgG, and the saturation concentration was used as the work of biotin-labeled goat anti-mouse IgG (BT-Ab) concentration.

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Abstract

The invention discloses a tree-like marker including nucleic acid, fluorescence molecule and protein / compound; a method for preparing the tree-like molecular; and an immunity detection reagent kit for forming the tree-like marker. The fluorescence molecule and the nucleic acid used as vector are combined into a signal marker in non-embedded manner, the signal marker and the protein or compound are bonded in non-covalent bond to from a tree-like structure; the label method is used for all immunity tests (tumor marker, communicable disease, hormone, heart disease, food contaminant, environmental pollutant, etc.), can obviously increase signal molecular number in immunity detection, and greatly improve detection sensitivity.

Description

Technical field [0001] The present invention relates to a label used in an antigen / antibody immunoassay and a preparation method thereof, and in particular, to a method for multi-labeling an immunoassay with a combination of nucleic acid and a fluorescent molecule. technical background [0002] Immunoassay methods have been widely used in the fields of life sciences, drug analysis, clinical diagnosis and environmental monitoring due to their high specificity, speed, simplicity and other advantages. Because the physical changes produced by the immune response are very weak and difficult to detect, substances or molecules that produce specific signals are usually labeled on antigens or antibodies, called markers. According to the different signals generated, the markers can be divided into radioisotopes, fluorescent dyes, electrochemical molecules, enzymes, chemiluminescence, electrochemiluminescence, etc. Among them, fluorescence detection has a wide range of applications and is a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64G01N33/533
Inventor 郭良宏张勤
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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