Human mycoplasma pneumoniae gold-marked silver-stained immunochromatographic assay kit and preparation method and application thereof

A technology for immunochromatographic detection and mycoplasma pneumoniae, which is applied in the field of medical detection, can solve the problems of inability to implement bedside detection, inconvenient clinical application, and insufficient convenient and fast time.

Active Publication Date: 2014-12-10
HUBEI UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the publicly reported methods for detecting Mp antigen are mainly double-antibody sandwich ELISA method, indirect immunofluorescence method, quantum dot-labeled immunochromatography method, etc., but these methods cannot be used for bedside detection, and need to be used on specific occasions. It is not only not convenient and fast enough but also takes a long time to detect with advanced instruments (such as microplate readers, fluorescence meters, etc.), and it is inconvenient for clinical application
[0005] Immunocolloidal gold chromatography is a micro-detection technology developed after radioimmunoassay and enzyme-linked immunoassay in recent years. It has a wide detection range, simple and fast operation (within 5-10 minutes), and can be used for bedside detection, etc. It is an ideal immunoassay method at present, but its clinical application is limited due to its low sensitivity
At present, there is no kit with high sensitivity and good stability for the detection of Mycoplasma pneumoniae antigen based on immunocolloidal gold chromatography

Method used

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  • Human mycoplasma pneumoniae gold-marked silver-stained immunochromatographic assay kit and preparation method and application thereof
  • Human mycoplasma pneumoniae gold-marked silver-stained immunochromatographic assay kit and preparation method and application thereof
  • Human mycoplasma pneumoniae gold-marked silver-stained immunochromatographic assay kit and preparation method and application thereof

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Experimental program
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Effect test

preparation example Construction

[0084] 1. Preparation of conjugated pads

[0085] (1) Preparation and purification of recombinant P1-His and P30-His fusion proteins:

[0086] Bioinformatics analysis was carried out on human Mycoplasma pneumoniae membrane proteins P1 and P30 to obtain the peptides with the most abundant antigenic epitopes in their extracellular domains; to find their corresponding gene coding sequences, according to the codon preference in Escherichia coli, After codon optimization, enzyme cleavage sites were introduced at the 5' and 3' ends respectively, and the entire gene sequence was chemically synthesized, and marked as p1 and p30 at the same time; see the sequence list for the sequence; the two gene sequences According to the method of molecular biology, respectively cloned into the expression vector pET-28a (+) and then transformed into Escherichia coli to express the recombinant P1-His, P30-His fusion protein; the recombinant P1-His fusion protein exists in the bacterium in a soluble ...

Embodiment 1

[0134] Embodiment 1 (preparation embodiment)

[0135] Conjugate pad preparation

[0136] (1) Preparation and purification of recombinant P1-His and P30-His fusion proteins

[0137] (1) Cloning of related genes

[0138]Bioinformatic analysis was performed on human Mycoplasma pneumoniae membrane proteins P1 and P30 (the accession numbers in the NCBI protein database are AAK92040 and ABR09215), respectively, to obtain the peptides with the most abundant antigenic epitopes in their extracellular conserved domains, and to find their The corresponding DNA coding sequence was codon-optimized according to the codon preference of Escherichia coli, and the restriction site NdeI was introduced at the 5' end, and the termination signal TAA and the restriction site XhoI were introduced at the 3' end, respectively. The whole gene sequence was synthesized (the whole sequence synthesis was completed by GenScript Biotechnology Co., Ltd., and the artificially synthesized gene fragments were a...

Embodiment 2

[0176] Embodiment 2 (preparation embodiment)

[0177] Preparation of sample pads

[0178] Prepare sample pad treatment solutions with different formulations, observe the release effect of colloidal gold-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 , sealed and dry at room temperature. Tests have proved that the use of the sample pad greatly improves the release rate of the colloidal gold-labeled antibody on the binding pad and achieves a better application effect.

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Abstract

The invention provides a human mycoplasma pneumoniae gold-marked silver-stained immunochromatographic assay kit and a preparation method and application thereof. The assay kit comprises a detection card and a silver-stained sensitivity-enhanced pad, wherein the detection card is composed of a bottom plate, a sample pad, an absorbent pad, a conjugate pad and a detection layer; the conjugate pad is coated with a colloidal gold-marked polyclonal antibody mixture of colloidal gold marked rabbit anti-human mycoplasma pneumoniae P1 protein and P30 protein; the detection layer is composed of a solid phase nitrocellulose membrane with a detection line and a quality control line; the detection layer is bonded on the bottom plate, the conjugate pad and the absorbent pad are partially overlapped with the detection layer respectively and are bonded with the detection layer and the bottom plate respectively; the sample pad and the conjugate pad are partially overlapped to be bonded with the conjugate pad and the bottom plate respectively; and the silver-stained sensitivity-enhanced pad consists of a AgNO3 pad and a restoring pad. The human mycoplasma pneumoniae gold-marked silver-stained immunochromatographic assay kit can effectively improve the detection sensitivity of the human mycoplasma pneumoniae, has the strong specificity and has the high application value in the aspects of clinical diagnosis of human mycoplasma pneumoniae, etiology identification, epidemiological investigation and the like.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a gold-labeled silver-stained immunochromatographic detection kit for human mycoplasma pneumoniae and a preparation method and application thereof. Background technique [0002] Mycoplasma pneumoniae (Mycoplasma pneumoniae, Mp) is the pathogen of human mycoplasma pneumonia, mainly transmitted through droplets, with an incubation period of 2-3 weeks, and the incidence rate is highest in adolescents. The incidence of Mp infection in children with pneumonia is as high as 10-30%. In recent years, it has gradually become one of the main pathogens of children with respiratory diseases. The disease can easily lead to respiratory infections such as pharyngitis and tonsillitis, and may even cause multiple organ injuries such as meningitis, hepatitis, and myocarditis at the same time, and may even lead to death in severe cases. [0003] Because the symptoms of Mp infection are s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/532G01N33/56933
Inventor 胡征杨波
Owner HUBEI UNIV OF TECH
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