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Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling

A Mycoplasma pneumoniae, magnetic separation technology, applied in the field of medical testing, can solve the problems of no clinical application, low positive rate, low content of human Mycoplasma pneumoniae, etc.

Active Publication Date: 2015-12-30
湖北诺美华抗体药物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the publicly reported methods for detecting human Mycoplasma pneumoniae antigens are mainly double-antibody sandwich ELISA method, quantum dot labeling immunochromatography method, indirect immunofluorescence method, etc., but these methods have high sensitivity, but due to the clinical The content in specimens such as throat swabs is extremely low, resulting in a low positive rate of clinical detection, and has not been used in clinical practice at present

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  • Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling
  • Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling

Examples

Experimental program
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Effect test

Embodiment 1

[0078] Example 1 Preparation of rabbit and mouse anti-human mycoplasma pneumoniae P1 protein polyclonal antibody IgG

[0079] (1) Preparation and purification of recombinant P1-His fusion protein

[0080] 1. Cloning of related genes

[0081] The human Mycoplasma pneumoniae membrane protein P1 (the accession number in the NCBI protein database is AAK92040) was analyzed by bioinformatics to obtain the most abundant epitope peptide in its extracellular conserved domain, find its corresponding DNA coding sequence, and then According to the codon preference of Escherichia coli, the codon was optimized, and the whole gene sequence was chemically synthesized after introducing the restriction site NdeI at the 5' end, the termination signal TAA and the restriction site XhoI at the 3' end (full sequence synthesis It was completed by GenScript Biotechnology Co., Ltd., and the artificially synthesized gene fragment was connected to the vector pUC57 at the time of delivery), denoted as p1...

Embodiment 2

[0096] Example 2 Preparation of Anti-human Mycoplasma pneumoniae Immune Nanomagnetic Beads

[0097] 1. Optimization of reaction conditions for anti-mycoplasma pneumoniae polyclonal antibody-coupled magnetic beads:

[0098] Using magnetic beads coupled with anti-mycoplasma pneumoniae polyclonal antibody as a solid-phase carrier, and quantum dot-labeled polyclonal antibody against mycoplasma pneumoniae P1 protein as a detection antibody, the human mycoplasma pneumoniae antigen was detected by the principle of double-antibody sandwich method, and the magnetic field was observed. Coupling of beads and polyclonal antibodies. A series of optimization options were carried out on the particle size of the magnetic beads, as well as the concentration of EDC / NHS activator, the concentration of conjugated antibody, the coupling time, and the type of blocking agent.

[0099] 1.1 Selection of magnetic bead size

[0100] Carboxylated magnetic beads with a particle size of 50nm, 180nm, 350n...

Embodiment 3

[0111] Example 3 Preparation of quantum dot-labeled anti-mycoplasma pneumoniae nanoprobes

[0112] 1. Optimization of the IgG reaction conditions for the nano carboxyl quantum dot-labeled mouse anti-human Mycoplasma pneumoniae P1 protein polyclonal antibody IgG:

[0113] 1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe

[0114] The pH of the phosphate buffer in the labeling reaction was set to 5, 6, 7, 8, and 9 respectively, and the fluorescence intensity of the labeled product was measured with a full spectrometer, and the influence of different pH values ​​on the coupling reaction was observed, and the quantum dot labeled polyclonal antibody was determined. The optimum pH for the reaction is 7.0-8.0. This experiment chooses pH7.4.

[0115] 1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes

[0116] Set the ratio of quantum dot molar concentration to polyclonal antibody concentrat...

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Abstract

The invention provides a method for detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling. The method comprises the following steps: (1) preparing anti-human chlamydia pneumoniae immune nano magnetic beads; (2) preparing quantum dot labelled anti-human chlamydia pneumoniae nanoprobes; (3) dissolving a sample to be detected with a PBST buffer solution, adding the anti-human chlamydia pneumoniae immune nano magnetic beads into the dissolved solution, carrying out magnetic separation after sufficient mixing and reaction, washing with the PBST buffer solution, adding the quantum dot labelled anti-human chlamydia pneumoniae nanoprobes into the obtained precipitate, carrying out magnetic separation after a reaction, washing with the PBST buffer solution, and detecting the fluorescence value with a fluorescence micro-plate reader. The method is accurate, fast, and high in sensitivity, and has very high practical value in the aspects of clinical diagnosis, etiological differentiation, epidemiological surveys and the like of human chlamydia pneumoniae.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a rapid detection method for detecting human mycoplasma pneumoniae antigen based on magnetic separation and quantum dot labeling, a detection kit, and a method for preparing and using the detection kit. Background technique [0002] Mycoplasma pneumoniae (Mycoplasmapneumoniae, Mp) is the pathogen of human mycoplasma pneumonia, mainly transmitted through droplets, with an incubation period of 2-3 weeks, and the incidence rate is highest in adolescents. The incidence of Mp infection in children with pneumonia is as high as 10-30%. In recent years, it has gradually become one of the main pathogens of children with respiratory diseases. The disease can easily lead to respiratory infections such as pharyngitis and tonsillitis, and may even cause multiple organ injuries such as meningitis, hepatitis, and myocarditis at the same time, and may even lead to death in severe cases...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
Inventor 胡征杨波董俊
Owner 湖北诺美华抗体药物技术有限公司
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