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Preparation method of latex microsphere immunochromatography test paper based on moraxella catarrhalis surface protein

A technology of surface protein and latex microspheres, applied in the direction of anti-bacterial immunoglobulin, immunoglobulin, chemical instruments and methods, etc., can solve problems such as difficult to meet rapid identification, affect the effect of amplification, and not suitable, and achieve The preparation cost is low, the shelf life is extended, and the effect of strong antigenicity

Active Publication Date: 2019-12-06
HUBEI UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]At present, the method for detecting the pathogen in the respiratory tract is mainly based on the traditional method, that is, the separation and identification method. Difficult to meet the needs of rapid identification; the PCR technology developed in recent years is a fast, sensitive, and specific technology, but at present this technology still relies on the pre-enrichment step of the traditional method, and the enrichment solution often There are also PCR inhibitors, which affect the effect of amplification
At the same time, this technology also requires professional testing equipment, which is not suitable for bedside testing

Method used

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  • Preparation method of latex microsphere immunochromatography test paper based on moraxella catarrhalis surface protein
  • Preparation method of latex microsphere immunochromatography test paper based on moraxella catarrhalis surface protein
  • Preparation method of latex microsphere immunochromatography test paper based on moraxella catarrhalis surface protein

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Effect test

Embodiment 1

[0060] Preparation of Antibody to Moraxella catarrhalis Surface Protein (M35+McaP):

[0061] 1.1) Cloning of Moraxella catarrhalis M35mac fusion gene

[0062] Obtain the peptides with the most abundant antigenic epitopes in the extracellular domain of the surface protein M35 and McaP of Moraxella catarrhalis (the accession numbers in the NCBI protein database are AY905613 and EF075940 respectively), find their gene coding sequences, and optimize their gene coding sequences , and link the two sequences with the coding sequence of the flexible linker peptide (ggsggsggsggs) to form a fusion gene. At the same time, the entire gene sequence was chemically synthesized after introducing the restriction site NdeI at the 5' end of the fusion gene, the termination signal TAA and the restriction site BamHI at the 3' end, and recorded as m35mac. The full sequence of the gene and the encoded amino acid sequence are shown in the sequence table. Specifically, the protein sequence encoded b...

Embodiment 2

[0091] Preparation of Moraxella catarrhalis surface protein (OlpA+OmpCD) antibody:

[0092] 2.1) Cloning of Moraxella catarrhalis Olpomp fusion gene

[0093] Obtain the peptides with the most abundant antigenic epitopes in the extracellular domain of the surface proteins OlpA and OmpCD of Moraxella catarrhalis (the accession numbers in the NCBI protein database are DQ996463 and AAA66180 respectively), find their gene coding sequences, and optimize their gene coding sequences , and the two sequences are connected with the coding sequence of the rigid linker peptide (eaaakeaaak) to form a fusion gene. At the same time, the entire gene sequence was chemically synthesized after introducing the restriction site NdeI at the 5' end of the fusion gene, the termination signal TAA and the restriction site BamHI at the 3' end, which was denoted as Olpomp. The full sequence of the gene and the encoded amino acid sequence are shown in the sequence table. Specifically, the protein sequenc...

Embodiment 3

[0123] Preparation of latex microsphere markers for antibodies against Moraxella catarrhalis surface protein (M35+McaP):

[0124] 3.1) Activation of latex microspheres

[0125] Take 1 mL of a solution of red carboxylated polystyrene latex microspheres (100 nm) with a concentration of 10%, and add 9 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (0.1mol / L MES, pH8.5 ) and mix well; use MES buffer to prepare 10mg / mL of N-hydroxysuccinimide (NHS) and 10mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt salt (EDC) solution;

[0126] Add 1mL NHS solution and 1mL EDC solution to the polystyrene latex microsphere (100nm) solution in turn, mix slowly at room temperature for 30 minutes, centrifuge at 19000g for 20 minutes after incubation, remove the supernatant, and use 10mL borax buffer ( 0.1mol / L Na 2 B 4 o 7 , pH8.5) resuspension, oscillation, ultrasonic treatment (ultrasonic breaker product model: YJ92-IIDN, power 50W, working time 2s, interval time ...

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Abstract

The invention relates to a preparation method of latex immunochromatography test paper for rapidly detecting moraxella catarrhalis. The test paper is composed of a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC plate, the combination pad is coated with an anti-moraxella catarrhalis surface protein polyclonal antibody coupled by colored latex microspheres, and the nitrocellulose membrane is coated with a detection line of an anti-moraxella catarrhalis surface protein polyclonal antibody and a quality control line of a goat anti-rabbit IgG antibody. When the added sample contains the moraxella catarrhalis, the moraxella catarrhalis and the latex-rabbit anti-moraxella catarrhalis surface protein polyclonal antibody firstly form a compound, the compound is captured when being migrated to a detection line coated with the moraxella catarrhalis surface protein polyclonal antibody under the capillary action, and the detection line shows a corresponding color, so that whether a sample contains the moraxella catarrhalis or not can be detected. The test paper has the advantages of rapidness, simplicity, high sensitivity and good specificity.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a preparation method of latex microsphere immunochromatographic test paper based on the surface protein of Moraxella catarrhalis. Background technique [0002] Moraxella catarrhalis (MC) was first discovered in 1896, when it was called Micrococcus catarrhalis, and later also known as Neisseria catarrhalis and catarrhalis bacteria (Branhamella catarhalis). In the past, Moraxella catarrhalis was considered to be a normal resident bacterium of the upper respiratory tract without pathogenicity to humans. However, research in the past 20 years has found that this bacterium can not only cause upper respiratory tract infections in children and the elderly, but also an important pathogenic bacteria that can cause lower respiratory tract infections in adults. The 3rd most common pathogenic bacteria, second only to Haemophilus influenzae and Streptococcus pneumoniae, and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K16/12C12N15/62C12N15/70G01N33/543G01N33/558G01N33/569G01N33/68
CPCC07K14/212C07K16/1214C07K2319/00C12N15/70G01N33/54313G01N33/558G01N33/56911G01N33/68
Inventor 杨波张秋胡征王毅
Owner HUBEI UNIV OF TECH
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