Magnetic separation and multicolor quantum dot marking-based method and kit for quickly detecting anti-human chlamydia pneumoniae IgM and IgG antibodies simultaneously

A Chlamydia pneumoniae and magnetic separation technology, applied in the field of medical testing, can solve problems such as long time, increased testing costs, increased labor intensity and operational errors

Active Publication Date: 2014-12-10
HUBEI UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The specificity and sensitivity of the cold agglutination test are the lowest, so its clinical diagnostic value is not great; although the gold-labeled immunoblot method and the gold-labeled immunochromatography method are simple and quick to operate, their sensitivity is slightly low, and they are not suitable for those with low antibody levels. Patients may miss the diagnosis; the preparation and operation of gelatin agglutination test reagents are cumbersome and require professional operators
Enzyme-linked immunosorbent assay has the advantages of specificity and sensitivity, and has been used to check various antibodies or antigens. However, this method has the following problems: (1) There are many steps in each batch of tests

Method used

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  • Magnetic separation and multicolor quantum dot marking-based method and kit for quickly detecting anti-human chlamydia pneumoniae IgM and IgG antibodies simultaneously
  • Magnetic separation and multicolor quantum dot marking-based method and kit for quickly detecting anti-human chlamydia pneumoniae IgM and IgG antibodies simultaneously
  • Magnetic separation and multicolor quantum dot marking-based method and kit for quickly detecting anti-human chlamydia pneumoniae IgM and IgG antibodies simultaneously

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Expression, purification and renaturation of recombinant M98-His fusion protein

[0075] 1. Cloning of related genes

[0076] Bioinformatics analysis was performed on the 98KD membrane protein of Chlamydia pneumoniae (its accession number in the NCBI protein database is CAA04672), and the peptide with the most abundant antigenic epitope in its extracellular conserved domain was obtained, and its corresponding DNA coding sequence was found. At the same time, the whole gene sequence was chemically synthesized 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 whole sequence synthesis was completed by GenScript Biotechnology Co., Ltd. The synthetic gene fragment was connected to the vector pUC57), which was denoted as M98. The full sequence of its gene is shown in the sequence listing. Specifically, the protein sequence encoded by the M98 gene is 130-305aa of the...

Embodiment 2

[0088] Example 2 Preparation of Anti-human Chlamydia pneumoniae Antibody Capturing Nano Magnetic Beads

[0089] 1. Optimization of reaction conditions for recombinant M98-His fusion protein coupled to magnetic beads:

[0090] Using magnetic beads coupled with recombinant M98-His fusion protein as a solid phase carrier, and quantum dot-labeled mouse anti-human IgM monoclonal antibody as a detection antibody, detect the positive serum of anti-human Chlamydia pneumoniae IgM antibody, and observe the interaction between the magnetic beads and the recombinant protein. Coupling situation. 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.

[0091] 1.1 Selection of magnetic bead size

[0092] Select carboxyl nano-magnetic beads with a particle size of 50nm, 180nm, 350nm, 1150nm, and 3μm, add PBS bu...

Embodiment 3

[0104] Example 3 Preparation of anti-human IgM and IgG nanoprobes labeled with multicolor quantum dots respectively

[0105] 1. Optimization of the reaction conditions for nanocarboxyl quantum dot-labeled mouse anti-human IgM monoclonal antibody:

[0106] 1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe

[0107] 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 monoclonal antibody was determined. The optimum pH for the reaction is 7.0-8.0. This experiment chooses pH7.4.

[0108] 1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes

[0109] Set the ratio of quantum dot molar concentration to monoclonal antibody concentration to 1:1, 1:2,...

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Abstract

The invention discloses a magnetic separation and multicolor quantum dot marking-based method and a kit for quickly detecting anti-human chlamydia pneumoniae IgM and IgG antibodies simultaneously. The kit is composed of nanometer magnetic anti-human chlamydia pneumoniae antibody capturing beads, a multicolor quantum dot marked anti-human IgM antibody nanometer probe, a multicolor quantum dot marked anti-human IgG antibody nanometer probe, quality control products and a PBST buffer solution, wherein the quality control products comprise positive quality control products and negative quality control products; the positive quality control products are positive anti-human chlamydia pneumoniae IgM and IgG antibodies of a human chlamydia pneumoniae infected person and are positive serums, and the negative quality control products are negative anti-human chlamydia pneumoniae IgM and IgG and are negative human serums. The method has the advantages of simplicity, quickness and high sensitivity, and can detect the anti-human chlamydia pneumoniae IgM and IgG antibodies simultaneously.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a detection method and detection kit for rapid co-detection of anti-human pneumoniae IgM and IgG antibodies based on magnetic separation and multicolor quantum dot labeling, and the preparation and use of the detection kit method. Background technique [0002] Chlamydia pneumoniae (Cpn), as an important respiratory pathogenic microorganism of the genus Chlamydia, was officially named by Grayston et al. in the mid-1980s, and has been extensively studied by researchers in the subsequent time. It is only known that humans are the host of the pathogen, and the infection may be transmitted from person to person through respiratory secretions. Children under the age of 5 are rarely infected, and children over the age of 8 and young people are susceptible to infection, especially in places where people gather, such as families, schools, and barracks. Serological epidemiologic...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/533
CPCG01N33/533G01N33/56927G01N33/577
Inventor 杨波胡征
Owner HUBEI UNIV OF TECH
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