Vascular hemophilia factor detection reagent, preparation method and application thereof

[0051] Example 1

[0052] The preparation method of the von Willebrand factor detection reagent in this embodiment includes the following steps:

[0053] (1) Preparation of R1: First prepare MES buffer with a concentration of 50 mM, and then add 0.5% by mass volume percentage NaCl and 0.08% by mass volume percentage Triton X-100, and adjust pH 7.2 after mixing .

[0054] (2) Preparation of R2: The preparation of 150nm latex microsphere system includes the following steps: using 100mL of EDC solution with a mass volume percentage of 2% and 100mL of 150nm latex microspheres with a mass volume percentage of 5% at 37°C constant temperature shaker Shake for 25 minutes, centrifuge the activated microspheres at 3000 rpm for 10 minutes, discard the supernatant, and wash 5 times with pH 6.5-7.0 MES buffer; then add 0.005% by mass and volume of mouse anti-human von Willebrand factor monomer The cloned antibody was mixed and incubated at 37°C for 5 hours. After incubation, it was washed 5 times with pH 6.5-7.0 MES buffer; then pH 6.5-7.0 containing 0.5% bovine serum albumin MES buffer was added for blocking for 2 hours ; Centrifuge and discard the supernatant, wash 3 times with pH 7.0 MES buffer, use 100 mL pH 7.0 containing 0.5% by mass and volume of NaCl and 0.5% by mass and volume of BSA MES buffer ultrasound Resuspend and store at 4°C until use.

[0055] The preparation of the 80nm latex microsphere system includes the following steps: mixing the mouse anti-human von Willebrand factor monoclonal antibody with a mass volume percentage of 0.005% and 80nm latex microspheres with a mass volume percentage of 5% at room temperature. After incubating for 80 minutes, add 100 mL of EDC solution with a mass volume percentage of 5%, incubate for 100 minutes, and then wash the antibody-coated microspheres for 6 times; add a 0.5% mass volume percentage of BSA solution at room temperature Block for 100min, centrifuge to remove the supernatant, wash the microspheres with deionized water 4-6 times, use 100mL pH7.0 MES buffer containing 0.5% by mass and volume of NaCl and 0.5% by mass and volume of BSA Resuspend by ultrasound and store at 4°C until use.

[0056] R2 is a mixture of 150nm latex microsphere system and 80nm latex microsphere system in a volume ratio of 1:1.

[0057] Example 2

[0058] The preparation method of the von Willebrand factor detection reagent in this embodiment includes the following steps:

[0059] (1) Preparation of R1: First prepare MES buffer with a substance concentration of 10 mM, then add 0.1% by mass volume percentage of NaCl and 0.01% by mass volume percentage of Triton X-100, and adjust pH 6.8 after mixing .

[0060] (2) Preparation of R2: The preparation of 150nm latex microspheres system includes the following steps: using 100mL of EDC solution with a mass volume percentage of 1% and 100mL of 150nm latex microspheres with a mass volume percentage of 15% at 37°C constant temperature shaker Shake for 30 minutes, centrifuge the activated microspheres at 3000 rpm for 10 minutes, discard the supernatant, and wash 4 times with pH6.5-7.0 MES buffer; then add mouse anti-human von Willebrand factor monomer with a mass volume percentage of 0.001% The cloned antibody was mixed and incubated at 37°C for 5 hours, and then washed with pH6.5-7.0 MES buffer once after incubation; then added with pH6.5-7.0 MES buffer containing 0.1% bovine serum albumin by mass and volume to block 1 -2h; centrifuge to discard the supernatant, wash 2-3 times with pH 7.0 MES buffer, use 100 mL pH 7.0 containing 0.1% by mass and volume of NaCl and 0.1% by mass and volume of BSA Resuspend in MES buffer by ultrasound and store at 4°C until use.

[0061] The preparation of the 80nm latex microsphere system includes the following steps: mixing the mouse anti-human von Willebrand factor monoclonal antibody with a mass volume percentage of 0.001% and the 80nm latex microspheres with a mass volume percentage of 15% at room temperature. After incubating for 100 minutes, add 100 mL of EDC solution with a mass volume percentage of 1%, incubate for 150 minutes, and then wash the antibody-coated microspheres 4 times; add a 0.1% mass volume percentage of BSA solution at room temperature Seal for 150 minutes, centrifuge to remove the supernatant, wash the microspheres with deionized water 5 times, and ultrasonically resuspend the microspheres with 100mL pH7.0 MES buffer containing 0.1% by mass and volume of NaCl and 0.1% by mass and volume of BSA. Suspend and store at 4°C until use.

[0062] R2 is a mixture of 150nm latex microsphere system and 80nm latex microsphere system in a volume ratio of 1:1.

[0063] Example 3

[0064] The preparation method of the von Willebrand factor detection reagent in this embodiment includes the following steps:

[0065] (1) Preparation of R1: First prepare MES buffer with a concentration of 200 mM, and then add 3% NaCl and 1% Triton X-100 by mass volume percentage. After mixing, adjust pH 7.4 .

[0066] (2) Preparation of R2: The preparation of 150nm latex microsphere system includes the following steps: use 100mL of EDC solution with a mass volume percentage of 10% and 100mL of 150nm latex microspheres with a mass volume percentage of 1% at 37°C constant temperature shaker Shake for 20 minutes, centrifuge the activated microspheres at 3000 rpm for 10 minutes, discard the supernatant, and wash 5 times with pH 6.5-7.0 MES buffer; then add the mouse anti-human von Willebrand factor monomer with a mass volume percentage of 0.05% The cloned antibody was mixed and incubated at 37°C for 5 hours. After incubation, washed 5 times with pH 6.5-7.0 MES buffer; then added pH 6.5-7.0 containing 3% bovine serum albumin MES buffer to block 1.5 h; Discard the supernatant by centrifugation, wash twice with pH 7.0 MES buffer, and use 100 mL pH 7.0 MES buffer containing 3% by mass and volume of NaCl and 3% by mass and volume of BSA Resuspend by ultrasound and store at 4°C until use.

[0067] The preparation of the 80nm latex microsphere system includes the following steps: mixing the mouse anti-human von Willebrand factor monoclonal antibody with a mass volume percentage of 0.05% and the 80nm latex microspheres with a mass volume percentage of 1% at room temperature After incubating for 60 minutes, add 100 mL of EDC solution with a mass volume percentage of 1%, incubate for 60 minutes, and then wash the antibody-coated microspheres 4 times; add a 3% mass volume percentage of BSA solution at room temperature Seal for 60 minutes, centrifuge to remove the supernatant, wash the microspheres with deionized water 6 times, and ultrasonically reproduce with 100 mL pH7.0 MES buffer containing 3% by mass and volume of NaCl and 3% by mass and volume of BSA. Suspend and store at 4°C until use.

[0068] R2 is a mixture of 150nm latex microsphere system and 80nm latex microsphere system in a volume ratio of 1:1.