Anti-human mycoplasma pneumonia P30 protein antibody and immunochromatographic kit using antibodoy

A technology of Mycoplasma pneumoniae and immunochromatography, applied in the field of biomedicine, can solve the problems of no natural protein structure, low titer, and low antibody specificity

Active Publication Date: 2016-08-17
HUBEI UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation method is simple and the cost is low, but the P30 protein expressed by genetic engineering has an inclusion body structure and does not have a natural protein structure, and the antibody prepared by it has defects such as low specificity and low titer

Method used

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  • Anti-human mycoplasma pneumonia P30 protein antibody and immunochromatographic kit using antibodoy
  • Anti-human mycoplasma pneumonia P30 protein antibody and immunochromatographic kit using antibodoy
  • Anti-human mycoplasma pneumonia P30 protein antibody and immunochromatographic kit using antibodoy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Preparation of rabbit anti-human Mycoplasma pneumoniae P30 protein antibody

[0076] The preparation method of rabbit anti-human Mycoplasma pneumoniae P30 protein antibody is as follows:

[0077] 1) After structural biology analysis and related experimental research, the 14 amino acids at positions 142-155 of the human Mycoplasma pneumoniae P30 protein (GenBank sequence number ABR09215.1) were selected as the linear antigen table for preparing rabbit anti-human Mycoplasma pneumoniae P30 protein antibodies The amino acid sequence of this segment is AHAEAEVEPAPQPV, and this sequence is named P30Linear;

[0078] 2) After adding a cysteine ​​to the N-terminus of the amino acid sequence P30Linear described in step 1), synthesize and purify the polypeptide with an automatic polypeptide synthesizer. The purified polypeptide is coupled with the carrier protein KLH to form a P30Linear-KLH composite protein ;

[0079] 3) Emulsify the compound protein synthesized in step 2), an...

Embodiment 2

[0085] Example 2 Preparation and application of immunochromatographic kit based on quantum dot labeling technology

[0086] 1. Quantum dot labeled antibody AbP30Linear

[0087] Add 0.4nmol carboxyl water-soluble quantum dots and 800nmol carbodiimide (EDC) to the microcentrifuge tube, and dilute the volume to 1ml with MES buffer (10.66g / L MES, 0.74g / L EDTA pH 7.4). Mix the solution in the ground and react at 37°C for 5 minutes, then add 0.34 mg of the antibody AbP30Linear prepared in Example 1, and react for 2 hours in the dark. Add single-end amino polyethylene glycol (PEG2000-NH2) to a final concentration of 1% (m / v), block the unreacted activated carboxyl sites and continue to react for 1h in the dark. After the reaction, the sample was centrifuged in an ultrafiltration tube (with a molecular weight cutoff of 100k), and centrifuged at 6500g for 5 minutes to a volume of 200ul. The ultrafiltration sample was transferred to a common EP tube and centrifuged to remove agglomeration ...

Embodiment 3

[0111] Example 3 Preparation and application of immunochromatographic kit based on colloidal gold labeling technology

[0112] 1. Colloidal gold labeled antibody AbP30Linear

[0113] a. Preparation of 30nm colloidal gold

[0114] Take a siliconized 250ml Erlenmeyer flask, add 99ml ultrapure water, add 1ml 1% (m / v) HAuCl4 solution to it and mix well, heat in an oil bath and stir until boiling. Quickly add 2 ml of 1% (m / v) trisodium citrate aqueous solution to it, and the solution continues to boil for 10 minutes (the solution changes from blue to red during this process). Stop heating, let the solution cool to room temperature naturally, and then add ultrapure water to make up to 100ml.

[0115] b. Colloidal gold labeled antibody AbP30Linear

[0116] 1) Take a siliconized 50ml Erlenmeyer flask, add 10ml of the colloidal gold solution prepared in step a, and add 240ul 0.2mol / L K to the gold solution 2 CO 3 Adjust the pH to 8.5;

[0117] 2) Add the antibody AbP30Linear to the colloidal go...

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Abstract

The invention relates to an anti-human mycoplasma pneumonia P30 protein antibody and an immunochromatographic kit for detecting human mycoplasma pneumonia by using the antibody. The anti-human mycoplasma pneumonia P30 protein antibody is an antibody for recognizing a linear epitope consisting of amino acids at 142-155 loci of a human mycoplasma pneumonia P30 protein; a sequence number of the human mycoplasma pneumonia P30 protein in GenBank is ABR09215.1; the amino acid sequences at 142-155 loci of the human mycoplasma pneumonia P30 protein are AHAEAEVEPAPQPV. The rabbit anti-human mycoplasma pneumonia P30 protein antibody provided by the invention has the characteristics of good specificity, high purity and titer and low preparation cost.

Description

Technical field [0001] The invention belongs to the field of biomedical technology, and relates to an anti-human Mycoplasma pneumoniae P30 protein antibody and an immunochromatographic kit for detecting human Mycoplasma pneumonia using the antibody. Background technique [0002] Mycoplasma pneumonia (M. Pneumonia) is the pathogen of human Mycoplasma pneumonia. The pathological changes of mycoplasma pneumonia are mainly interstitial pneumonia, sometimes complicated by bronchial pneumonia, called primary atypical pneumonia. It is mainly transmitted by droplets, with an incubation period of 2 to 3 weeks, and the incidence is highest among adolescents. The clinical symptoms are general respiratory symptoms such as headache, sore throat, fever, and cough, which can occur all year round, but mostly in autumn and winter. 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 respiratory disea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12G01N33/569G01N33/558
CPCC07K16/1253
Inventor 胡征董俊杨波
Owner HUBEI UNIV OF TECH
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