Natural antigen P1 for detecting anti-mycoplasma pneumoniae antibody as well as preparation method and application of natural antigen P1
A technology of Mycoplasma pneumoniae and natural antigen is applied in the field of biological detection, which can solve the problems of long time for stable cell expression, low protein expression, instability between batches, etc., and achieves easy expansion of production, low preparation cost, and mass production. Effect
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Embodiment 1
[0025] The preparation method of the natural antigen P1 of detection human mycoplasma pneumoniae antibody comprises the following steps:
[0026] (1) Collection of Mycoplasma pneumoniae: Take the inactivated human Mycoplasma pneumoniae antigen culture solution, centrifuge at 16,000 rpm for 0.5 h at 4°C to obtain Mycoplasma pneumoniae bacteria, wash twice with 10 mM PBS washing solution, and centrifuge at 4°C, 16,000 rpm Centrifuge for 0.5h, discard the supernatant, repeat washing twice, and the precipitate obtained is Mycoplasma pneumoniae;
[0027] (2) Freeze-thaw crushing treatment: first prepare a lysate containing 10 mM PBS, 5 mM Tcep, 2.5 mM EDTA, a final concentration of 2% arginine, and a final concentration of 2% trehalose, and adjust the pH of the lysate to 7.6; then put Mycoplasma pneumoniae into the lysate, mix well, incubate in a water bath at 37°C for 0.5 h, freeze at -20°C for 12 h, rethaw, centrifuge at 16,000 rpm for 0.5 h at 4°C, and discard the supernatant ,...
Embodiment 2
[0031] The preparation method of the natural antigen P1 of detection human mycoplasma pneumoniae antibody comprises the following steps:
[0032] (1) Collection of Mycoplasma pneumoniae: Take the inactivated human Mycoplasma pneumoniae antigen culture solution, centrifuge at 10,000 rpm for 2 h at 4°C to obtain Mycoplasma pneumoniae cells, wash with 20 mM PBS washing solution for 3 times, and centrifuge at 4°C, 10,000 rpm Centrifuge for 2 h, discard the supernatant, repeat washing once, and the precipitate obtained is Mycoplasma pneumoniae;
[0033] (2) Freeze-thaw crushing treatment: first prepare a lysate containing 20 mM PBS, 0.5 mM Tcep, 0.5 mM EDTA, a final concentration of 1% arginine, and a final concentration of 1% trehalose, and adjust the pH of the lysate to 7.6; then put Mycoplasma pneumoniae into the lysate, mix well, incubate in a water bath at 37°C for 2 hours, freeze at -20°C for 2 hours and rethaw, centrifuge at 10,000 rpm for 2 hours at 4°C, discard the superna...
Embodiment 3
[0037] The preparation method of the natural antigen P1 of detection human mycoplasma pneumoniae antibody comprises the following steps:
[0038] (1) Collection of Mycoplasma pneumoniae: Take the inactivated human Mycoplasma pneumoniae antigen culture medium, centrifuge at 12000 rpm for 1 h at 4°C to obtain Mycoplasma pneumoniae cells, wash once with 15 mM PBS washing solution, centrifuge at 4°C, 12000 rpm Centrifuge for 1 h, discard the supernatant, repeat washing twice, and the precipitate obtained is Mycoplasma pneumoniae;
[0039] (2) Freeze-thaw crushing treatment: first prepare a lysate whose components are 15 mM PBS, 1 mM Tcep, 1 mM EDTA, a final concentration of 5% arginine, and a final concentration of 5% trehalose, and adjust the pH of the lysate to 7.6; then put Mycoplasma pneumoniae into the lysate, mix well, incubate in a water bath at 37°C for 1 hour, freeze at -20°C for 4 hours and rethaw, centrifuge at 12,000 rpm for 1 hour at 4°C, discard the supernatant, The...
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