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Moraxella catarrhalis ELISA detection kit and preparation method based on Moraxella catarrhalis surface protein antibody

A surface protein and protein technology, applied in the field of biotechnology and infectious disease diagnosis research, can solve problems such as false positives, long time, difficult to meet rapid identification, etc.

Active Publication Date: 2021-04-06
HUBEI UNIV OF TECH
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  • Summary
  • 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
In addition, due to the sensitivity of the technique, false positives are often
At the same time, this technology also requires professional testing equipment, which is not suitable for bedside testing

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Preparation of polyclonal antibodies against Moraxella catarrhalis M35 and OlpA protein:

[0039] 1.1) Cloning of Moraxella catarrhalis M35olp fusion gene

[0040] Obtain the peptides with the most abundant antigenic epitopes in the extracellular domain of the surface protein M35 and OlpA of Moraxella catarrhalis (the accession numbers in the NCBI protein database are AY905613 and DQ996463 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 the restriction site NdeI was introduced into the 5' end of the fusion gene, the termination signal TAA and the restriction site BamHI were introduced into the 3' end, which was denoted as M35olp. The full sequence of the gene and the encoded amino acid sequence are shown in the sequence table. Spec...

Embodiment 2

[0070] Preparation of polyclonal antibodies against Moraxella catarrhalis McaP and OmpCD proteins:

[0071] 1.1) Cloning of the Mcapomp fusion gene of Moraxella catarrhalis

[0072] Obtain the peptides with the most abundant antigenic epitopes in the extracellular domain of the surface proteins McaP and OmpCD of Moraxella catarrhalis (the accession numbers in the NCBI protein database are EF075940 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 whole 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 Mcapomp. The full sequence of the gene and the encoded amino acid sequence are shown in the sequence table. Specifica...

Embodiment 3

[0101] Moraxella catarrhalis Elisa detection kit

[0102] 1. Composition of Moraxella catarrhalis Elisa Detection Kit

[0103] ELISA plate coated with polyclonal antibody against Moraxella catarrhalis M35 and OlpA protein, polyclonal antibody against Moraxella catarrhalis McaP and OmpCD protein, Moraxella catarrhalis positive control, negative control, washing solution, enzyme label The secondary antibody, enzyme chromogenic substrate and stop solution together constitute the Moraxella catarrhalis Elisa detection kit.

[0104] (1) ELISA plates coated with polyclonal antibodies against Moraxella catarrhalis M35 and OlpA protein

[0105] Anti-Moraxella catarrhalis M35 and OlpA protein polyclonal antibody (prepared in Example 1) was diluted to a concentration of 10 μg / mL with PBS buffer, and coated with a 96-well EIA high-efficiency binding enzyme plate ( Model: Corning costar2592), coated at 37°C for 2 hours. After taking it out, wash the plate three times with 250 μL washing...

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PUM

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Abstract

The invention relates to an Elisa detection kit for rapidly detecting Moraxella catarrhalis and a preparation method thereof. The kit includes: ELISA plates coated with polyclonal antibodies against Moraxella catarrhalis M35 and OlpA proteins, polyclonal antibodies against Moraxella catarrhalis McaP and OmpCD proteins, positive control and negative control for Moraxella catarrhalis , washing solution, enzyme-labeled secondary antibody, enzyme chromogenic substrate and stop solution. The kit of the present invention jointly detects four specific surface proteins of Moraxella catarrhalis, greatly improves the sensitivity, specificity and repeatability of Moraxella catarrhalis detection, and can be used for non-diagnosis of Moraxella catarrhalis sex testing and research work.

Description

technical field [0001] The invention belongs to the field of biotechnology and infectious disease diagnosis research, and relates to an Elisa detection kit, in particular to a Moraxella catarrhalis Elisa detection kit and a preparation method based on the Moraxella catarrhalis surface protein antibody. 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...

Claims

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Application Information

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