Human Haemophilus influenza quantum dot immunochromatography detection card, preparation method and application thereof

A technology for Haemophilus influenzae and Haemophilus bacillus is applied in the field of medical detection, which can solve the problems of long cell culture time, complicated operation steps, and missed detection.

Active Publication Date: 2016-01-13
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

However, this method has obvious defects such as complicated operation steps and long cell culture time, so it is not suitable for clinical application.
More importantly, the capsule swelling test and the like are limited to the identification of capsulated strains, while the non-capsulated type (NTHi) is completely missed.

Method used

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  • Human Haemophilus influenza quantum dot immunochromatography detection card, preparation method and application thereof
  • Human Haemophilus influenza quantum dot immunochromatography detection card, preparation method and application thereof
  • Human Haemophilus influenza quantum dot immunochromatography detection card, preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0076] 1. Preparation of conjugated pads

[0077] (1) Preparation and purification of recombinant P6-His fusion protein:

[0078] Bioinformatics analysis was carried out on the human Haemophilus influenzae P6 protein to obtain the peptide with the most abundant antigenic epitope in the extracellular domain of the human Haemophilus influenzae P6 protein, and to find its corresponding gene sequence; 5 of the two gene sequences Restriction sites were introduced at the 'end and 3' end respectively, and the whole gene sequence was chemically synthesized respectively, and marked as P6 at the same time. See the sequence listing for its gene sequence. The gene sequence was cloned into the expression vector pET-28a(+) according to the conventional method, and then the recombinant P6-His fusion protein was expressed. The fusion protein exists in the genetically engineered bacterium in the form of soluble expression. Purify the recombinant P6-His fusion protein in the genetically engi...

Embodiment 1

[0117] Embodiment 1 (preparation embodiment)

[0118] Conjugate pad preparation

[0119] (1) Preparation and purification of recombinant P6-His fusion protein

[0120] 1. Cloning of related genes

[0121] Bioinformatic analysis was performed on the surface protein P6 of human Haemophilus influenzae (its 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 to 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, 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. Specifica...

Embodiment 2

[0147] Embodiment 2 (preparation embodiment)

[0148] Preparation of sample pads

[0149] Prepare sample pad treatment solutions with different formulations, observe the release effect of quantum dot-labeled antibodies, and optimize through multiple orthogonal experiments to obtain the optimal sample pad treatment solution formulation (that is, described in the present invention). Take a piece of glass cellulose membrane, soak it in the sample pad treatment solution for at least 3 hours, then place it in a biological safety cabinet at 37°C, ventilate and dry it, and cut it into a size of 4cm*2.5cm / strip to prepare the sample pad , Store in airtight and dry place at 25°C. Tests have proved that the use of the sample pad greatly improves the release rate of the quantum dot-labeled antibody on the binding pad and achieves a better application effect.

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Abstract

The invention provides a human Haemophilus influenza quantum dot immunochromatography detection card, a preparation method and an application thereof. The detection card includes a base board, a sample pad, a water absorption pad, a combination pad and a detection layer. An anti-human Haemophilus influenza nano probe marked by quantum dots coats the detection layer. The detection layer is formed by a solid-phase nitrocellulose film having a detection line and a quality control line, wherein a mouse-anti-human Haemophilus influenza P6 protein polyclonal antibody coats the detection line and an anti-rabbit IgG coats the detection line. The detection layer is bonded to the base board. The combination pad and the water absorption pad are respectively disposed above the two ends of the detection layer, and then are partially overlapped with the detection and are respectively bonded to the detection layer and the base board. The sample pad is disposed above the combination pad, and then is partially overlapped with the combination pad and is respectively bonded to the combination pad and the base board. The detection card is simple in operations, is quick in detection and allows quantitative and high-sensitive detection.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a human Haemophilus influenzae quantum dot immunochromatographic detection card and a preparation method and application thereof. 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 disability or even death in children. Hi is divided into six serotypes: a, b, c, d, e, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/531G01N33/533
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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