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
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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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