Human vWF-A2 DuoSet ELISA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. -Working dilutions should be prepared and used immediately.	-A standard curve should be generated for each set of samples assayed. -It is suggested to start Reagent Diluent optimization for serum and plasma samples by using PBS supplemented with 10-50% animal serum. -Do not use buffers with animal serum to reconstitute or dilute the Detection Antibody or Streptavidin-HRP B.

Upstream tips
-Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. -Working dilutions should be prepared and used immediately.
Protocol tips
-A standard curve should be generated for each set of samples assayed. -It is suggested to start Reagent Diluent optimization for serum and plasma samples by using PBS supplemented with 10-50% animal serum. -Do not use buffers with animal serum to reconstitute or dilute the Detection Antibody or Streptavidin-HRP B.

Publication protocol

Serum levels of 18 markers of inflammation and fibrosis, selected based on previous association with LC and its prognosis (Table 2) were measured in duplicate using commercially available reagents by enzyme- immunoassay (EIA; all proteins except vWF: R&D Systems, Minneapolis, MN, USA; vWF: DakoCytomation, Glostrup, Denmark) in a 384-format using the combination of a SELMA (Jena, Germany) pipetting robot and a BioTek (Winooski, VT, USA) dispenser/washer (EL406). Primary and secondary antibody concentrations were used according to manufacturer (Coating 1–4 μg/mL; secondary 0.2–2 μg/mL). Assay volume was 25 μL and coating was performed in phosphate buffered saline (PBS, SCBT, Heidelberg, Germany). Subsequent assay buffer was 1% bovine serum albumin (VWR, Radnor, PA, USA) in PBS while sample diluent was assay buffer with 25% heat inactivated fetal calf serum (Gibco, Thermo Fisher Scientific, Waltman, MA, USA). Wash buffer was PBS with 0.05% tween20 and 3 wash cycles were included per step. Samples were incubated overnight at 4 °C. Absorption was read at 450 nm with wavelength correction set to 540 nm using an EIA plate reader (Synergy H1 Hybrid, Biotek, Vinooski, VT, USA). Intra- and inter-assay coefficients of variation were < 10% for all ass.

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Manufacturer protocol

Download the product protocol from R&D Systems for Human vWF-A2 DuoSet ELISA below.

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