Anti-human Streptococcus pneumoniae fam2 family pspa protein antibody and immunochromatographic kit using the antibody

A streptococcus pneumoniae and immunochromatography technology, applied in the field of biomedicine, can solve the problems of low titer, low specificity, and low purity

Active Publication Date: 2019-07-19
HUBEI UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its preparation method is simple and low in cost, but it has defects such as low specificity, low titer, and low purity.

Method used

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  • Anti-human Streptococcus pneumoniae fam2 family pspa protein antibody and immunochromatographic kit using the antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Preparation of two kinds of rabbit anti-human Streptococcus pneumoniae fam2 family PspA protein antibodies

[0079] The preparation methods of two kinds of rabbit anti-human Streptococcus pneumoniae fam2 family PspA protein antibodies are as follows:

[0080] 1) After structural biology analysis and related experimental research, a short sequence composed of 14 amino acids at positions 34-47 and 14 amino acids at positions 274-287 of the fam2 family PspA protein of human Streptococcus pneumoniae (GenBank sequence number WP_054380072.1) was selected. The peptides are respectively used as two linear epitopes for preparing rabbit anti-human Streptococcus pneumoniae fam2 family PspA protein antibodies. The two amino acid sequences are SPQVVEKSSLEKKY and KLLDSLDPEGKTQD respectively, and these two sequences are named F2P1 and F2P2 respectively;

[0081] 2) After adding a cysteine ​​to the C-terminal of F2P1 and the N-terminal of F2P2 in the amino acid sequence descr...

Embodiment 2

[0088] Example 2 Preparation and Application of Immunochromatography Kit Based on Quantum Dot Labeling Technology

[0089] 1. Quantum dot-labeled antibody AbF2P1

[0090] Add 0.4nmol carboxyl water-soluble quantum dots and 800nmol carbodiimide (EDC) to the microcentrifuge tube successively, make the volume to 1ml with MES buffer (10.66g / L MES, 0.74g / L EDTA pH 7.4), keep Mix the solution with ground, react at 37°C for 5 minutes, then add 0.4 mg of the antibody AbF2P1 prepared in Example 1, and react in the dark for 2 hours. Add single-terminal amino polyethylene glycol (PEG2000-NH2) to a final concentration of 1% (m / v), block the unreacted activated carboxyl site, and continue to react in the dark for 1h. The reacted sample was centrifuged with an ultrafiltration tube (molecular weight cut-off 100k), centrifuged at 6500g for 5min to a volume of 200ul, transferred the ultrafiltered sample to an ordinary EP tube, and centrifuged to remove aggregates (10000g, 3min). Add the sup...

Embodiment 3

[0111] Example 3 Preparation and Application of Immunochromatography Kit Based on Colloidal Gold Labeling Technology

[0112] 1. Colloidal gold-labeled antibody AbF2P1

[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 therein and mix evenly, heat in an oil bath and stir until boiling. 2ml of 1% (m / v) trisodium citrate aqueous solution was quickly added thereto, and the solution continued to boil for 10 min (the solution turned from blue to red during this process). Stop heating, let the solution cool down to room temperature naturally, then add ultrapure water to it to make up to 100ml.

[0115] b. Colloidal gold-labeled antibody AbF2P1

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

[0117] 2) Add the antibody AbF2P1 into the co...

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Abstract

The invention relates to antibodies for resisting human streptococcus pneumoniae fam2 family PspA protein and an immunochromatographic kit applying the antibodies to detection on human streptococcus pneumoniae. The antibodies for resisting the human streptococcus pneumoniae fam2 family PspA protein are used for recognizing two linear antigenic epitopes composed of 34-47 amino acids and 274-287 amino acids of the human streptococcus pneumoniae fam2 family PspA protein respectively, and the sequence number of the human streptococcus pneumoniae fam2 family PspA protein in GenBank is WP_054380072.1; the sequences of the 34-47 amino acids and the 274-287 amino acids of the human streptococcus pneumoniae fam2 family PspA protein are SPQVVEKSSLEKKY and KLLDSLDPEGKTQD respectively. The two rabbit antibodies for resisting the human streptococcus pneumoniae fam2 family PspA protein have the advantages of being good in specificity, high in purity and titer and low in preparation cost.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to an anti-human Streptococcus pneumoniae fam2 family PspA protein antibody and an immunochromatographic kit using the antibody to detect human Streptococcus pneumoniae. Background technique [0002] Streptococcus pneumoniae (Sp) is an important pathogen of respiratory tract infection in children. It enters the respiratory tract mainly through droplets, parasitizes the nasopharynx of the human body, or invades parts of the human body that are not easy to remove, causing a series of diseases, such as lobar pneumonia, meningitis, bronchitis, otitis media, etc. It is the leading pathogen with high morbidity and mortality in all age groups worldwide. Among them, in developing countries, infants, the elderly, and immunocompromised populations are particularly serious. Streptococcus pneumoniae was first isolated from patient sputum in France and the United States by Louis Pasteur and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12G01N33/569G01N33/558G01N33/532
CPCC07K16/1275G01N33/532G01N33/558G01N33/56944G01N2333/3156
Inventor 胡征董俊杨波
Owner HUBEI UNIV OF TECH
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