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A validated sandwich ELISA for the detection of von Willebrand factor in rabbit plasma
Author(s) -
Smith Brendon W.,
Strakova Jana,
King Jennifer L.,
Tu Michael J.,
Erdman John W.,
O'Brien William D.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.542.2
Subject(s) - von willebrand factor , horseradish peroxidase , chemistry , detection limit , antibody , microbiology and biotechnology , chromatography , coagulation , biomarker , platelet , fibrinogen , microtiter plate , immunology , enzyme , biochemistry , medicine , biology
Von Willebrand Factor (vWF) is a multimeric plasma protein important for platelet plug formation. We are interested in evaluating vWF as a biomarker of endothelial damage in atherosclerotic rabbits that were exposed to cardiovascular ultrasound. We developed a sensitive and reproducible sandwich enzyme‐linked immunosorbent assay (ELISA) for detection of vWF in rabbit plasma using commercially available antibodies and reagents. Briefly, 96‐well microtiter plates were coated with antibody generated in mice against the n‐terminus of vWF overnight followed by blocking in 0.2% BSA in PBS for 2 hrs. Plates were incubated with rabbit plasma for 2 hrs and exposed first to a goat anti‐vWF “sandwich” antibody and then a donkey anti‐goat IgG antibody conjugated to horseradish peroxidase, each for 1 hr. An OPD substrate solution was added to the plate for 30 minutes. The chromogenic reaction was stopped by addition of 0.5 N H 2 SO 4 and the plate was read at 490 nm. Several aspects of the assay were evaluated including non‐specific signal, intra‐ and inter‐assay precision, lower limit of detection, percent recovery after standard addition, and interference of other plasma components. This ELISA provides sensitive and reproducible measurements of rabbit plasma vWF, which is an important biomarker for cardiovascular ultrasound research. [Supported by NIH R37EB002641]