Method and kit for rapidly detecting human haemophilus influenzae based on magnetic separation and quantum dot labelling

A technology of Haemophilus influenzae and Haemophilus bacillus, which is applied in the field of medical detection, can solve the problems of missed detection, unsuitable for clinical application, complicated operation steps, etc.

Active Publication Date: 2016-01-06
湖北诺美华抗体药物技术有限公司
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  • Claims
  • Application Information

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Problems solved by technology

However, this method has obvious defects such as complicated operation steps and long cell culture time, so it is not suitable for clinical application.
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  • Method and kit for rapidly detecting human haemophilus influenzae based on magnetic separation and quantum dot labelling
  • Method and kit for rapidly detecting human haemophilus influenzae based on magnetic separation and quantum dot labelling

Examples

Experimental program
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Effect test

Embodiment 1

[0081] Example 1 Preparation of rabbit and mouse anti-human Haemophilus influenzae membrane protein P6 polyclonal antibody IgG

[0082] (1) Preparation and purification of recombinant P6-His fusion protein

[0083] 1. Cloning of related genes

[0084] 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 sho...

Embodiment 2

[0098] Example 2 Preparation of anti-human Haemophilus influenzae immune nano-magnetic beads

[0099] 1. Optimization of reaction conditions for anti-Haemophilus influenzae polyclonal antibody coupled to magnetic beads:

[0100] Using magnetic beads coupled with anti-Haemophilus influenzae membrane protein P6 polyclonal antibody as a solid phase carrier, quantum dot-labeled anti-human Haemophilus influenzae membrane protein P6 polyclonal antibody as a detection antibody, through the principle of double antibody sandwich method Detect human Haemophilus influenzae antigen, and observe the coupling between 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.

[0101] 1.1 Selection of magnetic bead size

[0102] Carboxylated magnetic beads with a particle ...

Embodiment 3

[0113] Example 3 Preparation of quantum dot-labeled anti-human Haemophilus influenzae nanoprobes

[0114] 1. Optimization of IgG reaction conditions for nano carboxy quantum dot-labeled mouse anti-human Haemophilus influenzae membrane protein P6 polyclonal antibody IgG:

[0115] 1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe

[0116] 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.

[0117] 1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes

[0118] Set the ratio of quantum dot molar concentration to polyclonal antibody co...

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Abstract

The invention provides a method for detecting a human haemophilus influenzae antigen based on magnetic separation and quantum dot labelling. The method includes the steps: (1) preparing anti-human haemophilus influenzae immune nano magnetic beads; (2) preparing quantum dot labelled anti-human haemophilus influenzae nano probe; and (3) after dissolving a to-be-tested sample in a PBST buffer solution, adding the anti-human haemophilus influenzae immune nano magnetic beads to the dissolved solution, fully mixing, carrying out a reaction, then carrying out magnetic separation, washing with a PBST buffer solution, adding the quantum dot labelled anti-human haemophilus influenzae nano probe to the obtained precipitate, carrying out a reaction, then carrying out magnetic separation, washing with a PBST buffer solution, and then detecting a fluorescent value by using a fluorescence microplate reader. The accurate, rapid and high-sensitivity method for detecting human haemophilus influenzae is established, and has quite high practical value in clinical diagnosis, etiology identification and epidemiological investigation of human haemophilus influenzae.

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 human Haemophilus influenzae (Hi) antigen based on magnetic separation and quantum dot labeling, and the preparation and use of the detection kit method. 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 world, which can cause disabi...

Claims

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Application Information

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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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