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Streptococcus pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof

A streptococcus pneumoniae and immunochromatography technology, applied in the field of medical testing, can solve problems such as complicated operation steps, high quality requirements for sample materials to be tested, false positives, etc.

Active Publication Date: 2016-02-10
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

Neither immunofluorescence method nor immunoenzyme method can perform one-step detection, and both have the disadvantages of complicated operation steps, professional operation, long detection time (more than 2 hours), and high cost.
The PCR method is fast, sensitive, and specific, and is an important method for studying Streptococcus pneumoniae infection. However, due to the high requirements for experimental equipment and operation of PCR, and the possibility of false positives, it cannot be used as a commonly used clinical diagnosis method in my country.
At present, the method for detecting human Streptococcus pneumoniae antigen is mainly the colloidal gold method to detect its C polysaccharide antigen, but the sensitivity of the colloidal gold method is low, and the quality requirements of the sample materials to be tested are high. There are defects such as cross-reactivity

Method used

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  • Streptococcus pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof
  • Streptococcus pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof
  • Streptococcus pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof

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preparation example Construction

[0082] 1. Preparation of conjugated pads

[0083] (1) Preparation and purification of recombinant PspA1-His and PspA2-His fusion proteins:

[0084] Bioinformatics analysis was performed on the Fam1PspA and Fam2PspA proteins of human Streptococcus pneumoniae, and the peptides with the most abundant antigenic epitopes in the extracellular domains of the Fam1PspA and Fam2PspA proteins of Human Streptococcus pneumoniae were respectively obtained, and their corresponding gene sequences were found; The 5' end and 3' end of the sequence were respectively introduced with enzyme cutting sites, and the whole gene sequence was chemically synthesized respectively, and marked as PspA1 and PspA2 at the same time. See the sequence listing for their gene sequences. The two gene sequences were respectively cloned into the expression vector pET-28a(+) according to conventional methods, and then recombinant PspA1-His and PspA2-His fusion proteins were expressed. These two fusion proteins both ...

Embodiment 1

[0128] Embodiment 1 (preparation embodiment)

[0129] Conjugate pad preparation

[0130] (1) Preparation and purification of recombinant PspA1-His and PspA2-His fusion proteins

[0131] 1. Cloning of related genes

[0132] 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 ful...

Embodiment 2

[0159] Embodiment 2 (preparation embodiment)

[0160] Preparation of sample pads

[0161] 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 streptococcus pneumoniae quantum dot immunochromatographic assay detection card and a preparing method and application thereof. The detection card comprises a bottom board, a sample pad, a water absorbing pad, a combining pad and a detection layer. Streptococcus pneumoniae resisting nanometer probes labeled with quantum dots envelop the combining pad. The detection layer is formed by a solid-phase nitrocellulose membrane with a detection line and a quality control line. Anti-mouse streptococcus pneumoniae PspA protein polyclonal antibodies envelop the detection line. Anti-rabbit IgG envelops the quality control line. The detection layer is bonded to the bottom board. The combining pad and the water absorbing pad are arranged on the upper portions of the two ends of the detection layer respectively, partially overlapped with the detection layer and then bonded to the detection layer and the bottom board. The sample pad is arranged on the combining pad, partially overlapped with the combining pad and then bonded to the combining pad and the bottom board. The streptococcus pneumoniae quantum dot immunochromatographic assay detection card has the advantages of being easy and convenient to operate, fast in detection, capable of achieving quantification, high in sensitivity and the like.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a human Streptococcus pneumoniae quantum dot immunochromatographic detection card and its preparation method and application. 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 particularly serious. Streptococcus pneumoniae was first isolated from patient sputum in France and the United States by Pasteur (Louis Paste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/532
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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