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A von Willebrand factor ristocetin cofactor ELISA kit

An enzyme-linked immunosorbent reagent, ristocetin technology, applied in the direction of biological testing, color/spectral characteristic measurement, material inspection products, etc., to achieve the effect of reducing reagent cost, high sensitivity, and less plasma consumption

Inactive Publication Date: 2011-12-14
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] But, there is no commercialized vWF:Rcof-ELISA diagnostic kit so far in the prior art

Method used

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  • A von Willebrand factor ristocetin cofactor ELISA kit
  • A von Willebrand factor ristocetin cofactor ELISA kit
  • A von Willebrand factor ristocetin cofactor ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Preparation of hybridoma cell line SZ-151.

[0038] Using normal human mixed platelets as the immunogen, 8-week-old female Balb / c mice were routinely immunized three times (each interval was 4 weeks), and the presence of monoclonal antibodies in the serum of immunized animals was detected by enzyme-linked immunosorbent assay (ELISA) and concentration. After immunization, select mice that produce antiserum with a sufficiently high concentration (the antibody titer in the serum is above 1:20,000), separate the spleen of the animal, and prepare a spleen cell suspension.

[0039] The obtained mouse splenocytes were combined with appropriate myeloma ( SP2 / 0), after screening and identification by biochemical and immunological techniques such as ELISA and Western blotting, the hybridoma with high antibody secretion level was selected, and the sustainable generation and secretion of anti-von Willebrand factor were prepared. A hybridoma cell line for monoclonal ant...

Embodiment 2

[0041] Example 2: Purification of monoclonal antibody SZ-151.

[0042] Prepare monoclonal antibody SZ-151 ascites according to conventional techniques, dialyze in PH8.0, 0.1M PBS buffer, centrifuge at 8000rpm, take about 1ml of ascites supernatant and pass through the column (affinity chromatography column Protein G-Sepharose 4B) to make each IgG is fully combined with protein G, eluted with 0.1M pH2.8 glycine-hydrochloric acid buffer, and the IgG peaks of each monoclonal antibody are collected. The collected IgG was concentrated with PEG (molecular weight about 20,000), dialyzed in 0.01MPBS for 4 hours, and the protein content was measured with 280nm, and the SZ-151-IgG was frozen in a low-temperature refrigerator for later use.

Embodiment 3

[0043] Example 3: SZ-151 IgG solid-phase coated microtiter plate.

[0044] Dilute the anti-GPIbα monoclonal antibody SZ-151-IgG with coating buffer (0.1M, pH9.6 carbonic acid buffer), add 10 μg / mL to the wells of a 96-well polystyrene microplate, 100 μl / well, Overnight in a humid box at 4°C; the next day, wash 3 times with PBS-0.05% Tween, block with 250 μl / well of blocking solution (2% BSA-PBS), and overnight in a wet box at 4°C; the next day, wash 3 times as above before use Or freeze at -20°C.

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PUM

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Abstract

The invention discloses an ELISA kit for von Willebrand factor ristocetin cofactor, comprising: a solid-phase coated antibody ELISA plate, a detection antibody labeled with horseradish peroxidase, and a standard product, ristocetin, recombinant GPIbαH1-V289 fragment-Fc fusion protein, sample dilution buffer, chromogenic solution, stop solution, wherein the coating antibody is anti-platelet membrane glycoprotein Ibα monoclonal antibody SZ- 151; the detection antibody is rabbit anti-human VWF polyclonal antibody; the standard product is mixed standard plasma. When the kit is used to detect vWF:RCof, it has good repeatability and sensitivity, and the test results have good correlation with vWF:Ag and vWF:CBA.

Description

technical field [0001] The invention belongs to enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) technology, in particular to a test kit for detecting the activity of von Willebrand factor ristocetin cofactor, using anti-platelet glycoprotein (glycoprotein, GP ) Ibα monoclonal antibody (monoclonal antibody, mAb) SZ-151 is used as a coating antibody to capture the binding eukaryotic expression of recombinant human GPIbα amino-terminal His 1 -Val 289 Fragmented -Fc fusion protein (rfGPIbα H1-V289-Fc), recaptured von Willebrand factor (VWF) in plasma, and then detected VWF ristocetin cofactor with enzyme label detector active. Background technique [0002] Von Willebrand factor (vWF) is a large molecular weight multimeric glycoprotein with adhesion function, which plays an important role in the initial hemostasis process. It can not only bind to platelet receptor glycoprotein (GP) Ib / IX / V, GPⅡb / Ⅲa, but also bind to extracellular matrix (ECM) such ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531G01N21/31G01N33/96
Inventor 赵益明阮长耿季顺东杨剑锋
Owner SUZHOU UNIV
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