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

A Streptococcus pneumoniae and magnetic separation 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: 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 Streptococcus pneumoniae igm and IgG antibodies based on magnetic separation and multicolor quantum dot labeling
  • Method and kit for rapid co-examination of anti-human Streptococcus pneumoniae igm and IgG antibodies based on magnetic separation and multicolor quantum dot labeling
  • Method and kit for rapid co-examination of anti-human Streptococcus pneumoniae igm and IgG antibodies based on magnetic separation and multicolor quantum dot labeling

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1 Preparation and purification of recombinant human Streptococcus pneumoniae PspA protein

[0077] 1. Cloning of related genes

[0078] Bioinformatic analysis was performed on the Fam1PspA and Fam2PspA proteins of human Streptococcus pneumoniae (the accession numbers in the NCBI protein database are AAF27703 and AAF27712), respectively, to obtain the peptides with the most abundant antigenic epitopes in their extracellular domains, and to find their corresponding 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. , the artificially synthesized gene fragments were connected to the vector pUC57 at the time of delivery), which were denoted as PspA1 and PspA2. The full sequence of its gene is shown in the sequence listing. Wherein, the ...

Embodiment 2

[0088] Example 2 Preparation of anti-human Streptococcus pneumoniae antibody capture nano-magnetic beads

[0089] 1. Optimization of reaction conditions for recombinant human Streptococcus pneumoniae PspA protein-coupled magnetic beads:

[0090] Using the magnetic beads coupled with the recombinant human Streptococcus pneumoniae PspA protein as the solid phase carrier, the mouse anti-human IgM monoclonal antibody labeled with quantum dots as the detection antibody, detect the anti-human Streptococcus pneumoniae 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, an...

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 method and a kit for performing quick co-detection on anti-Sp (Streptococcus pneumoniae) IgM (Immunoglobulin M) and IgG (Immunoglobulin G) antibodies based on magnetic separation and multi-color quantum dot labeling. The kit consists of anti-Sp antibody capturing nano magnetic beads with an anti-Sp 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 substance comprise a positive quality control substance and a negative quality control substance; the positive quality control substance is serum, in which anti-Sp IgM and IgG antibodies of Sp infected people are respectively positive; the negative quality control substance is serum, in which anti-Sp 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-Sp 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-human Streptococcus pneumoniae IgM and IgG antibodies based on magnetic separation and multicolor quantum dot labeling, as well as the preparation and testing kit of the detection kit. Instructions. Background technique [0002] Human Streptococcus pneumoniae (Streptococcuspneumoniae, Sp) is an important pathogen of respiratory tract infection in children. It enters the respiratory tract mainly through droplets, parasitizes the nasopharynx of the human body, or invades parts of the human body that are not easy to remove, causing a series of diseases, such as lobar pneumonia, meningitis, bronchitis, otitis media, etc. It is the leading pathogen with high morbidity and mortality in all age groups worldwide. Among them, in developing countries, infants, the elderly, and immunocompromised populations are ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/569G01N21/64
CPCG01N21/6486G01N33/56944G01N33/577G01N2333/3156
Inventor 胡征杨波董俊
Owner HUBEI UNIV OF TECH
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