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

A Chlamydia pneumoniae, magnetic separation technology, applied in the field of medical detection, can solve the problems of long cell culture time, unsatisfactory sensitivity, specificity and repeatability, and inability to directly identify Chlamydia pneumoniae

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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Problems solved by technology

However, the PCR experiment has special requirements for the laboratory, the sample processing, amplification and detection requirements are strict, and it is prone to false positives, so it cannot be used as a commonly used clinical diagnostic method in my country.
[0005] Antigen detection methods include separation and culture of chicken embryo yolk sac and cell culture method (two kinds of cells, HL and Hep-2, are commonly used), and then use fluorescent-labeled or enzyme-labeled antibodies to detect Chlamydia in the specimen, but this method has complicated operation steps and cell culture Obvious defects such as long time, not suitable for clinical application
In recent years

Method used

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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

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Example Embodiment

[0082] Example 1 Preparation of rabbit and mouse anti-human Chlamydia pneumonia 98KD membrane protein polyclonal antibody IgG

[0083] (1) Preparation and purification of recombinant M98-His fusion protein

[0084] 1. Cloning of related genes

[0085] The 98KD membrane protein of human Chlamydia pneumoniae (the accession number in the NCBI protein database is CAA04672) was analyzed by bioinformatics, and the peptide fragment with the most abundant epitope in the extracellular conserved domain was obtained, and the corresponding DNA coding sequence was found. After introducing the restriction site NdeI at the 5'end of the sequence, the termination signal TAA and the restriction site XhoI at the 3'end, the complete gene sequence is chemically synthesized (the complete sequence synthesis is completed by GenScript Biotechnology Co., Ltd., and artificially synthesized upon delivery Attached to the vector pUC57), denoted as m98. The complete gene sequence is shown in the sequence table. ...

Example Embodiment

[0100] Example 2 Preparation of anti-human Chlamydia pneumoniae immune nano magnetic beads

[0101] 1. Optimization of reaction conditions for anti-human Chlamydia pneumoniae polyclonal antibody coupled to magnetic beads:

[0102] Using magnetic beads coupled with anti-human Chlamydia pneumoniae 98KD membrane protein polyclonal antibody as solid phase carrier, quantum dot-labeled anti-human Chlamydia pneumoniae 98KD membrane protein polyclonal antibody as detection antibody, detection of human Chlamydia pneumoniae by the principle of double antibody sandwich method Antigen, observe the coupling between magnetic beads and polyclonal antibody. A series of optimization selections were made for the particle size of the magnetic beads, the concentration of EDC / NHS activator, the concentration of coupling antibody, coupling time, and the type of blocking agent.

[0103] 1.1 Selection of magnetic bead size

[0104] Choose the carboxyl magnetic beads with particle size of 50nm, 180nm, 350nm,...

Example Embodiment

[0115] Example 3 Preparation of quantum dot-labeled anti-human Chlamydia pneumoniae nanoprobe

[0116] 1. Optimization of IgG reaction conditions for mouse anti-human Chlamydia pneumonia 98KD membrane protein polyclonal antibody labeled with nano-carboxyl quantum dots:

[0117] 1.1. Determination of the optimal labeling pH for carboxyl quantum dot labeled antibody probe

[0118] The pH of the phosphate buffer in the labeling reaction was set to 5, 6, 7, 8, 9 respectively. The fluorescence intensity of the labeled product was measured by a full-spectrometer, and the effect of different pH values ​​on the coupling reaction was observed. Quantum dot-labeled polyclonal antibodies were determined The optimal pH of the reaction is 7.0-8.0. This experiment chooses pH7.4.

[0119] 1.2 Determination of the optimal labeling amount of carboxyl quantum dot-labeled antibody probe

[0120] Set the ratio of the molar concentration of quantum dots to the concentration of polyclonal antibody to 1:1, 1...

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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 sample treating 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 a 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 and a detection kit for detecting human Chlamydia pneumoniae (Cpn) antigen based on magnetic separation and quantum dot labeling, as well as preparation and use methods of the detection kit. Background technique [0002] Chlamydiapneumoniae (Cpn), as an important respiratory pathogenic microorganism of the genus Chlamydia, has been officially named by Grayston et al. in the mid-1980s and has been extensively studied by researchers in the following period. It is only known that humans are the host of the pathogen, and the mode of infection may be spread from human to human through respiratory secretions. Children under 5 years of age are rarely infected. Children over 8 years of age and young people are susceptible to infection, especially in crowded areas such as homes, schools, and barracks. Seroepidemiological investigations have confirmed that at ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531G01N33/533
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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