Vascular hemophilia factor detection reagent, preparation method and application thereof

A hemophilia factor and detection reagent technology, applied in the field of clinical medical detection, can solve the problems of lack of reliability, cumbersome detection, low repeatability, etc., and achieve the effects of improving coupling efficiency, accurate test results, and stable detection

Inactive Publication Date: 2018-02-09
北京众驰伟业科技发展有限公司
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Problems solved by technology

However, this method is cumbersome to detect, has poo...
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Abstract

The invention provides a vascular hemophilia factor detection reagent, a preparation method and application thereof. The detection reagent includes two latex microsphere systems with different particle sizes. The vascular hemophilia factor detection reagent prepared through the preparation method is high in repeatability and accurate and rapid in detection, limiting influencing factors are fewer,results are reliable, and the detection reagent is low in cost and beneficial to clinical medical applications.

Application Domain

Disease diagnosisBiological testing

Technology Topic

ChemistryMicrosphere +3

Image

  • Vascular hemophilia factor detection reagent, preparation method and application thereof
  • Vascular hemophilia factor detection reagent, preparation method and application thereof
  • Vascular hemophilia factor detection reagent, preparation method and application thereof

Examples

  • Experimental program(7)

Example Embodiment

[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.

Example Embodiment

[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.

Example Embodiment

[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.

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