Method for detecting interaction of antigen and antibody by using FRET (Fluorescence Resonance Energy Transfer) technology

An antigen-antibody technology, applied in measuring devices, biological testing, material inspection products, etc., can solve the problems of operation errors affecting the experimental results, complicated work and long time, etc., to achieve clear chemical composition and dosage, fast detection time, and time-sensitive fast effect

Pending Publication Date: 2022-05-13
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Commonly used immunoassay methods include immunolabeling technology (enzyme-linked immunosorbent assay, ELISA), agglutination reaction, precipitation reaction, etc. Most of the technologies require repeated incubation / washing steps, and the antigen or antibody needs to be fixed and blocked, and the work is complicated and takes a long time , and prone to operational errors affecting the experimental results

Method used

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  • Method for detecting interaction of antigen and antibody by using FRET (Fluorescence Resonance Energy Transfer) technology
  • Method for detecting interaction of antigen and antibody by using FRET (Fluorescence Resonance Energy Transfer) technology
  • Method for detecting interaction of antigen and antibody by using FRET (Fluorescence Resonance Energy Transfer) technology

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Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Labeling the pretreatment-activated fluorescein group with the protein to be tested

[0028] 1. Prepare 20mMPBS (containing 15mMNacl) phosphate buffer: 1LNa2HPO4(12H2O) is alkaline and 1LNaH2PO4(2H2O) is acidic, mix them together, adjust the pH to 7.2, total 2L, add chlorination according to 2L*15mMNacl=0.03molNacl Sodium powder, filter the prepared solution through filter paper for later use.

[0029] 2. Pretreatment of rhodamine B fluorescein: take 1 mg of rhodamine B powder and dissolve it in 1 ml of 20 mMPBS (containing 15 mM Nacl) phosphate buffer saline and MES solution in a mixed solution. ml of RB solution, mixed according to the molar ratio RB:EDC:NHS=1:1.2:1.2, and activated at room temperature for 4 hours in the dark.

[0030]3. Rhodamine B and PCT antigen labeling: Take 1ml of the purified PCT antigen with a concentration of 1mg / ml and dialyze it once every 3 hours with 20mMPBS (containing 15mM Nacl) solution at 4°C, for a total of three times. ...

Embodiment 2

[0032] Embodiment 2: Sephadex G-25 molecular sieve screening purification

[0033] 1. Column packing: Pack the column overnight one day in advance, put the swollen dextran gel into a circular glass column with a diameter of 1.5 cm, the liquid height is about 12 cm, and settle overnight at 4°C.

[0034] 2. Column equilibration: Use 80-100ml of double distilled water to pass through the column to wash away the 75% ethanol solution, and then use 80-100ml of 20mMPBS (containing 15mM Nacl) phosphate solution to equilibrate the column.

[0035] 3. Sample loading: Avoid light during the sample loading process, and add the sample when the equilibrium solution in the column is flush with the top layer of the gel.

[0036] 4. Elution: Use the balance solution as the eluent to elute the sample, and the labeling is successful. It can be seen that after the eluent is added, the column is divided into three colors: upper, middle and lower. The upper layer is darker fluorescein, and the midd...

Embodiment 3

[0037] Embodiment 3: Multifunctional microplate reader detects fluorescence intensity

[0038] 1. Detection of FITC-PCT antibody labeling rate: Take 100ul of the screened FITC-PCT antibody solution in a cuvette, measure the absorbance at 495nm and 280nm, and calculate the F / P molar ratio. Formula: Molar F / P =[2.77×A495] / [A280-(0.35×A495)] calculation, the ratio is about 1, indicating higher efficiency.

[0039] 2. FRET technology to detect PCT antigen-antibody interaction: using a multi-functional microplate reader, add 100ulFITC-PCT antibody to a well in a 96-well plate, and detect the fluorescence spectrum and intensity at 495nm excitation and 530nm emission wavelength; in the 96-well plate Add 100ulFITC-PCT antibody and 100ulRB-PCT antigen solution to the second well, mix well, react at room temperature in the dark for 20min, and detect the fluorescence spectrum and intensity at 495nm excitation and 580nm emission wavelength.

[0040] 3. After statistical analysis of the f...

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Abstract

The invention belongs to the technical field of chemiluminescence biology, and particularly relates to a fluorescence resonance energy transfer technology which is applied to detection of antigen-antibody affinity interaction. The invention provides an FRET (Fluorescence Resonance Energy Transfer) detection technology which comprises the following steps: selecting fluorescein isothiocyanate (FITC) and Rhodamine B as fluorescent markers, pretreating the Rhodamine B and a coupling agent EDC/NHS to activate carboxyl groups, carrying out labeling reaction on fluorescein groups and antigens and antibodies, screening and purifying labeled samples through Sephadex G-25, reacting the labeled antigens and antibodies in an elisa plate, and detecting the fluorescence in the elisa plate. FRET technology is utilized to measure fluorescence intensity, and interaction is detected. The detection technology provided by the invention aims to establish a platform by utilizing a fluorescence labeling technology, is used for screening and detecting the interaction of the antigen and the antibody, is a convenient technology which does not need repeated incubation and washing circulation, can be directly used for reaction detection in a solution, and can be applied to the aspects of antibody preparation, immunotherapy research, protein function detection and the like.

Description

technical field [0001] The invention belongs to the technical field of chemiluminescent biology, in particular to a fluorescence resonance energy transfer technology, which is applied to the detection of antigen-antibody affinity interaction. Background technique [0002] Fluorescence resonance energy transfer (FRET) is a technology to detect whether there is a direct interaction between biological macromolecules in the nanometer distance in the living body. It is a kind of energy generated between two fluorescent molecules that are very close phenomenon of transfer. When the emission spectrum of the donor fluorescent molecule overlaps with the absorption spectrometer of the acceptor fluorescent molecule, and the distance between the two molecules is within 10nm, the fluorescence of the donor is quenched, while the fluorescence emitted by the acceptor is greatly enhanced, a phenomenon occurs. Nonradiative energy transfer. FRET technology is widely used, and it can detect p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/543G01N33/58
CPCG01N21/6428G01N33/543G01N33/582
Inventor 卢彦沈萍萍王秋萍
Owner NANJING UNIV
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