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Rapid detection method and kit for Moraxella catarrhalis based on magnetic separation and quantum dot labeling

A magnetic separation and quantum dot technology, applied in the field of medical detection, can solve problems such as complicated operation steps and long detection time

Active Publication Date: 2017-02-01
湖北诺美华抗体药物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is the "gold standard" for detecting Moraxella catarrhalis infection, but it has obvious defects such as complicated operation steps and long detection time, so it is not very suitable for clinical application

Method used

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  • Rapid detection method and kit for Moraxella catarrhalis based on magnetic separation and quantum dot labeling
  • Rapid detection method and kit for Moraxella catarrhalis based on magnetic separation and quantum dot labeling
  • Rapid detection method and kit for Moraxella catarrhalis based on magnetic separation and quantum dot labeling

Examples

Experimental program
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Effect test

Embodiment 1

[0083] Example 1 Preparation of Rabbit and Mouse Anti-Moraxella catarrhalis UspA1 Protein Polyclonal Antibody IgG

[0084] (1) Preparation and purification of recombinant UspA1-His fusion protein

[0085] 1. Cloning of related genes

[0086] Bioinformatics analysis was carried out on the surface protein UspA1 of Moraxella catarrhalis (the accession number in the NCBI protein database is AAF36416), to obtain the peptide with the most abundant antigenic epitope in its extracellular conserved domain, and to find its corresponding DNA code At the same time, 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 (the whole sequence synthesis was completed by GenScript Biotechnology Co., Ltd., and the delivery When the artificially synthesized gene fragment was connected to the vector pUC57), it was denoted as UspA1. The full sequence of its gene is shown i...

Embodiment 2

[0099] Example 2 Preparation of Anti-Moraxella catarrhalis Immune Nano Magnetic Beads

[0100] 1. Optimization of reaction conditions for anti-Moraxella catarrhalis polyclonal antibody coupled to magnetic beads:

[0101] Using magnetic beads coupled with anti-M. catarrhalis polyclonal antibody as a solid phase carrier, and quantum dot-labeled anti-M. Bacillus antigen, observe the coupling of magnetic 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.

[0102] 1.1 Selection of magnetic bead size

[0103] Select carboxyl magnetic beads with a particle size of 50nm, 180nm, 350nm, 1150nm, and 3 μm, add PBS buffer solution containing 4mg / ml EDC and 4mg / ml NHS to carry out the activation reaction, and then react with the rabbit anti-cardiac beads described in Examp...

Embodiment 3

[0114] Example 3 Preparation of quantum dot-labeled anti-Moraxella catarrhalis nanoprobes

[0115] 1. Optimization of IgG reaction conditions for nanocarboxyl quantum dot-labeled mouse anti-Moraxella catarrhalis UspA1 protein polyclonal antibody IgG:

[0116] 1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe

[0117] 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.

[0118] 1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes

[0119] Set the ratio of quantum dot molar concentration to polyclonal antibody concentration to 1:1, ...

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Abstract

The invention provides a method for detection of moraxella catarrhalis based on magnetic resolution and quantum dot labelling. The method comprises the following steps: (1) preparing anti-moraxella catarrhalis nano immunomagnetic beads; (2) preparing quantum dot labelled anti-moraxella catarrhalis nanoprobes; (3) dissolving a sample to be detected with a PBST buffer solution, adding the anti-moraxella catarrhalis 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-moraxella catarrhalis 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 identification, epidemiological surveys and the like of moraxella catarrhalis.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a rapid detection method and detection kit for detecting Moraxella catarrhalis (Mc) antigen based on magnetic separation and quantum dot labeling, as well as the preparation and testing kit for the detection kit. Instructions. Background technique [0002] Moraxella catarrhalis (Mc) was first discovered in 1896 and was called Micrococcus catarrhalis at that time, and later also known as Neisseria catarrhalis and Branham catarrhalis (Branhamella catarrhalis). Moraxella catarrhalis is a Gram-negative coccus, usually reniform diplococcus, occasionally tetrad, no flagella, no spores, and generally no capsule. Its nutritional requirements are not high, and it can grow on ordinary medium, aerobic, and the optimum growth temperature is 35°C. The diameter of the colony is 1-3 mm, smooth, off-white, opaque, and the whole colony is easy to scrape off from the culture medium. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/566G01N21/64
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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