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Moraxella catarrhalis quantum dot-immunochromatography detection card and preparation method and application thereof

A technology of immunochromatography and quantum dots, applied in the field of medical testing, can solve problems such as long detection time and complicated operation steps

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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is the "gold standard" for detecting Moraxella catarrhalis infection, but it has obvious defects such as complicated operation steps and long detection time, so it is not very suitable for clinical application

Method used

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  • Moraxella catarrhalis quantum dot-immunochromatography detection card and preparation method and application thereof

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Experimental program
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Effect test

preparation example Construction

[0078] 1. Preparation of conjugated pads

[0079] (1) Preparation and purification of recombinant UspA1-His fusion protein:

[0080] Bioinformatic analysis of Moraxella catarrhalis UspA1 protein was carried out to obtain the peptide with the most abundant antigenic epitope in the extracellular domain of Moraxella catarrhalis UspA1 protein, and to find its corresponding gene sequence; the 5' of the gene sequence Restriction sites were introduced at the end and 3' end respectively, and the whole gene sequence was chemically synthesized, and marked as UspA1 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 UspA1-His fusion protein was expressed. The fusion protein exists in the genetically engineered bacteria in the form of inclusion body expression. Purify the recombinant UspA1-His fusion protein in the genetically engineered bacter...

Embodiment 1

[0119] Embodiment 1 (preparation embodiment)

[0120] Conjugate pad preparation

[0121] (1) Preparation and purification of recombinant UspA1-His fusion protein

[0122] 1. Cloning of related genes

[0123] Bioinformatics analysis was performed on the surface protein UspA1 of Moraxella catarrhalis (its accession number in the NCBI protein database is AAF36416), 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 UspA1. The full sequence of its gene is shown in the sequence listing. Specif...

Embodiment 2

[0149] Embodiment 2 (preparation embodiment)

[0150] Preparation of sample pads

[0151] 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 moraxella catarrhalis (Mc) quantum dot-immunochromatography detection card and a preparation method and an application thereof. The detection card comprises a bottom plate, a sample pad, a water absorption pad, a conjugate pad and a detection layer, wherein the conjugate pad is coated by a quantum dot labelled anti-Mc nano-probe; the detection layer is formed by a solid-phase nitrocellulose membrane with a detection line and a quality control line; the detection line is coated by a mouse anti-Mc UspA1 protein polyclonal antibody; the quality control line is coated by anti-rabbit IgG (immunoglobulin); the detection layer is stuck to the bottom plate; the conjugate pad and the water absorption pad are respectively arranged above the two ends of the detection layer and are respectively stuck to the detection layer and the bottom plate after partially overlapping with the detection layer; the sample pad is arranged above the conjugate pad and is stuck to the conjugate pad and the bottom plate respectively after partially overlapping with the conjugate pad. The detection card has the advantages of simplicity and convenience in operation, high detection speed, capability of quantifying, high sensitivity and the like.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a Moraxella catarrhalis quantum dot immunochromatographic detection card and a preparation method and application thereof. Background technique [0002] Moraxella catarrhalis (Mc) was first discovered in 1896 and was called Micrococcus catarrhalis at that time, and later also known as Neisseria catarrhalis and Branhamella catarrhalis . Moraxella catarrhalis is a Gram-negative coccus, usually reniform diplococcus, occasionally tetrad, no flagella, no spores, and generally no capsule. Its nutritional requirements are not high, and it can grow on ordinary medium, aerobic, and the optimum growth temperature is 35°C. The diameter of the colony is 1-3 mm, smooth, off-white, opaque, and the whole colony is easy to scrape off from the culture medium. After a long period of culture, the colonies can be rough and granular, adhering to the surface of the medium. Produces oxida...

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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