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Method and kit for performing quick co-detection on anti-Mc (Moraxella catarrhalis) IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies based on magnetic separation and multi-color quantum dot labeling

A magnetic separation and quantum dot technology, applied in the field of medical detection, can solve the problems of inconvenient and fast detection, low specificity and sensitivity, and missed diagnosis of patients.

Active Publication Date: 2014-12-03
HUBEI UNIV OF TECH +1
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  • Abstract
  • Description
  • Claims
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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, such as adding samples, warming baths, and washing plates. It is cumbersome and takes a long time (2-4 hours in total); (2) The color components A and B need to be added separately during the detection, and the reading must be completed within the specified time, which increases labor intensity and operation to a certain extent. Possibility of mistakes; (3) This method cannot achieve simultaneous detection of IgM and IgG antibodies
Although individual detection of IgM and IgG antibodies and comprehensive analysis of the test results have no effect on the diagnosis of the disease, the operation process is cumbersome, the detection is not convenient and fast, and the cost of detection will increase compared with the joint detection

Method used

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  • Method and kit for performing quick co-detection on anti-Mc (Moraxella catarrhalis) IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies based on magnetic separation and multi-color quantum dot labeling
  • Method and kit for performing quick co-detection on anti-Mc (Moraxella catarrhalis) IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies based on magnetic separation and multi-color quantum dot labeling
  • Method and kit for performing quick co-detection on anti-Mc (Moraxella catarrhalis) IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies based on magnetic separation and multi-color quantum dot labeling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Expression, purification and renaturation of recombinant UspA1-His fusion protein

[0077] 1. Cloning of related genes

[0078] 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 in the sequence listing. Specifically, the protein sequence encoded by the UspA1 gene is 522-710aa...

Embodiment 2

[0088] Example 2 Preparation of Anti-Moraxella catarrhalis Antibody Capturing Nano Magnetic Beads

[0089] 1. Optimization of reaction conditions for recombinant UspA1-His fusion protein coupled to magnetic beads:

[0090] Using the magnetic beads coupled with the recombinant UspA1-His fusion protein as the solid phase carrier, the mouse anti-human IgM monoclonal antibody labeled with quantum dots as the detection antibody, detect the anti-Moraxella catarrhalis IgM antibody positive serum, observe the magnetic beads and recombinant protein coupling. 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 buffer containing 4mg / ml EDC and 4mg...

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 the molar concentration of quantum dots to the concentration of monoclonal antibo...

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Abstract

The invention discloses a method and a kit for performing quick co-detection on anti-Mc (Moraxella catarrhalis) IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies based on magnetic separation and multi-color quantum dot labeling. The kit consists of anti-Mc antibody capturing nano magnetic beads with an anti-Mc IgM and IgG antibody gathering function, anti-human IgM and IgG antibody nano probes labeled by multi-color quantum dots, quality control substances and a PBST buffering solution, wherein the quality control substances comprise a positive quality control substance and a negative quality control substance; the positive quality control substance is serum, in which anti-Mc IgM and IgG antibodies of Mc infected people are respectively positive; the negative quality control substance is serum, in which anti-Mc IgM and IgG are respectively negative. The kit and the method have the advantages of simplicity, quickness and high sensitivity, and can be used for carrying out synchronous detection on the anti-Mc IgM and IgG antibodies.

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-Moraxella catarrhalis IgM and IgG antibodies based on magnetic separation and multicolor quantum dot labeling, and the preparation of the detection kit and how to use it. 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 cultu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531G01N33/533
CPCG01N33/54326G01N33/54346G01N33/56911G01N2333/212G01N2333/22
Inventor 杨波胡征
Owner HUBEI UNIV OF TECH
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