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Homogeneous Fluorescence Detection of Serum Protein Based on Fluorescence Resonance Energy Transfer

A fluorescence resonance energy, homogeneous fluorescence technology, applied in the field of medical testing, can solve the problems of complex substances, poor stability, changing serum redox conditions, etc.

Active Publication Date: 2020-07-07
WUHAN LIFE ORIGIN BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the existing homogeneous immunoassay method based on fluorescence resonance energy transfer (FRET) detects serum samples, which has poor stability, and the substances in serum samples are very complex, which may contain various drugs or components that will change the redox conditions of serum , has a greater impact on the detection of samples in serum, and has a greater impact on the detection method of directly using antibody heavy and light chains and the antigen to be detected

Method used

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  • Homogeneous Fluorescence Detection of Serum Protein Based on Fluorescence Resonance Energy Transfer
  • Homogeneous Fluorescence Detection of Serum Protein Based on Fluorescence Resonance Energy Transfer
  • Homogeneous Fluorescence Detection of Serum Protein Based on Fluorescence Resonance Energy Transfer

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Effect test

Embodiment 1

[0018] The concentration determination of embodiment 1 C-reactive protein (CRP)

[0019] 1. Nanobody fluorescent labeling

[0020] (1) Fluorescence-labeled nanobody preparation Adjust the concentration of the nanobody solution to 20 mg / mL with physiological saline and carbonate buffer, mix with 1 / 10 of the total amount of buffer, and stir in an ice bath for 5-10 minutes. Add an appropriate amount of fluorescein AF488 into the nanobody solution gradually, and stir at 4°C for 12h-18h.

[0021] (2) Purification of fluorescently labeled antibodies Put the fluorescent antibody solution in a dialysis bag, dialyze with running tap water for 5 min, then dialyze with 0.01mol / L, pH 7.2PBS for 4h, pass through a DEAE cellulose column, and use 0.01mol / L , 0.05mol / L, and 0.14mol / L NaCl in PBS, and the labeled antibodies with F / P values ​​around 1.5 were collected and stored for future use.

[0022] 2. Quencher labeled antibody

[0023] The method is the same as the fluorescein-labeled a...

Embodiment 2

[0036] The concentration determination of embodiment 2 lipocalin (NGAL)

[0037] 1. Nanobody fluorescent labeling

[0038] (1) Fluorescence-labeled nanobody preparation Adjust the concentration of the nanobody solution to 20 mg / mL with physiological saline and carbonate buffer, mix with 1 / 10 of the total amount of buffer, and stir in an ice bath for 5-10 minutes. Add an appropriate amount of fluorescein to the nanobody solution gradually, and stir at 4°C for 12h to 18h.

[0039] (2) Purification of fluorescently labeled antibodies Put the fluorescent antibody solution in a dialysis bag, dialyze with running tap water for 5 min, then dialyze with 0.01mol / L, pH 7.2PBS for 4h, pass through a DEAE cellulose column, and use 0.01mol / L , 0.05mol / L, and 0.14mol / L NaCl in PBS, and the labeled antibodies with F / P values ​​around 1.5 were collected and stored for future use.

[0040] 2. Quencher labeled paired antibody

[0041] The method is the same as the fluorescein-labeled antibod...

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Abstract

The invention provides a method for detecting serum protein by homogeneous fluorescence detection on the basis of fluorescence resonance energy transfer. The method adopts nanobodies marked by fluorescent dye as energy donors, and adopts homogeneous nanobodies or paired nanobodies marked by a corresponding fluorescent quenching agent as energy receptors. The method provided by the invention has the beneficial effects that with adoption of the nanobodies, the stability is good, and the detection for samples in serum is basically not influenced; the nanobodies marked by the fluorescent dye are used as the energy donors, the homogeneous nanobodies or paired nanobodies marked by the corresponding fluorescent quenching agent are used as the energy receptors, and by adoption of a method of double-antibody sandwiching, the defects of low sensitivity and influence of oxidizing and reducing substances of a serum sample in the immunoassay method are effectively overcome, and the fastness and thesensitivity of the method are ensured.

Description

technical field [0001] The invention relates to the technical field of medical testing, in particular to a method for detecting serum protein with homogeneous fluorescence based on fluorescence resonance energy transfer. Background technique [0002] Immunoassay is a trace biological analysis method, which utilizes the high affinity between antigens and antibodies and the high measurability of markers used as probes to perform accurate quantitative analysis of trace substances in organisms, and has the advantages of simple operation, With the advantages of good specificity and high sensitivity, it has become an important means of research in biology, medicine, chemistry and other disciplines. Immunoassays can be divided into homogeneous immunoassays and heterogeneous immunoassays according to the physical state of the reaction system. Among the existing immunoassay methods, RIA, ELISA and CLIA are the most widely used heterogeneous immunoassay methods, but heterogeneous imm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N33/533
CPCG01N21/6486G01N33/533
Inventor 华权高沈鹤霄徐春雷
Owner WUHAN LIFE ORIGIN BIOTECH LTD
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