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Method and kit for rapid co-examination of anti-human Haemophilus influenzae igm and IgG antibodies based on magnetic separation and multicolor quantum dot labeling

A Haemophilus, magnetic separation technology, applied in the field of medical testing, can solve the problems of long time, increased labor intensity, operational errors, and inability to achieve simultaneous detection, etc.

Active Publication Date: 2016-01-06
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, 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 rapid co-examination of anti-human Haemophilus influenzae igm and IgG antibodies based on magnetic separation and multicolor quantum dot labeling
  • Method and kit for rapid co-examination of anti-human Haemophilus influenzae igm and IgG antibodies based on magnetic separation and multicolor quantum dot labeling
  • Method and kit for rapid co-examination of anti-human Haemophilus influenzae igm and IgG antibodies based on magnetic separation and multicolor quantum dot labeling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Preparation and purification of recombinant human Haemophilus influenzae P6 protein

[0076] 1. Cloning of related genes

[0077] Bioinformatic analysis of human Haemophilus influenzae membrane protein P6 (the accession number in the NCBI protein database is AAA24994) to obtain the peptide with the most abundant antigenic epitope in its extracellular conserved domain, and find its corresponding DNA coding sequence 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., upon delivery The artificially synthesized gene fragment is connected to the vector pUC57), which is denoted as P6. The full sequence of its gene is shown in the sequence listing. Specifically, the protein sequence encoded by the P6 gene is 48-153aa of the ...

Embodiment 2

[0086] Example 2 Preparation of anti-human Haemophilus influenzae antibody capture nano-magnetic beads

[0087] 1. Optimization of reaction conditions for recombinant P6-His fusion protein coupled to magnetic beads:

[0088] Magnetic beads coupled with recombinant human P6-His fusion protein were used as solid phase carrier, and mouse anti-human IgM monoclonal antibody labeled with quantum dots was used as detection antibody to detect the positive serum of anti-human Haemophilus influenzae IgM antibody. Conjugation of recombinant proteins. 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.

[0089] 1.1 Selection of magnetic bead size

[0090] Select carboxyl nano-magnetic beads with a particle size of 50nm, 180nm, 350nm, 1150nm, and 3μm, add PBS buffer containing 4mg / mlEDC and 4mg / mlNHS for ...

Embodiment 3

[0102] Example 3 Preparation of anti-human IgM and IgG nanoprobes labeled with multicolor quantum dots respectively

[0103] 1. Optimization of the reaction conditions for nanocarboxyl quantum dot-labeled mouse anti-human IgM monoclonal antibody:

[0104] 1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe

[0105] 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.

[0106] 1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes

[0107] 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 method and a kit for performing quick co-detection on anti-human Hi (Haemophilus influenzae) IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies based on magnetic separation and multi-color quantum dot labeling. The kit consists of anti-human Hi antibody capturing nano magnetic beads with an anti-human Hi 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-human Hi IgM and IgG antibodies of human Hi infected people are respectively positive; the negative quality control substance is serum, in which anti-human Hi 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-human Hi 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-Haemophilus influenzae 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] Haemophilus influenzae (Hi) is an important respiratory pathogenic microorganism that infects humans. This bacterium was discovered by Polish bacteriologist Dr. Feffer in an influenza plague in 1892, and it was discovered in the following time. Researchers have studied extensively. Only humans are known to be the host of the pathogen. The elderly and children with poor immunity are susceptible groups, especially infants and young children under 5 years old. Hi can cause pneumonia, conjunctivitis, otitis media, meningitis and bacteremia, etc., and at least 3 million severe cases occur every year in the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/569G01N21/64
CPCG01N33/533G01N33/54346G01N33/5695G01N33/577
Inventor 杨波胡征董俊
Owner HUBEI UNIV OF TECH
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