Human Angiopoietin-like 3 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

Maternal peripheral plasma samples were collected and stored in EDTA at -80°C prior to testing. Plasma samples were assessed (as indicated) for levels of Ang1, Ang2, Angiopoietin-Like 3 (AngptL3), Vascular Endothelial Growth Factor (VEGF-A), Factor D, Monocyte Chemoattractant Protein-1 (MCP-1), Interleukin-1 Beta (IL1B), Soluble fms-like tyrosine kinase 1 (sFlt-1), soluble Tumor Necrosis Factor Receptor 2 (sTNFR2), Placental Growth Factor (PGF), Macrophage Inflammatory Protein-1 Beta (MIP-1β), Leptin, Interleukin-18 Binding Protein (IL-18BP), soluble Intercellular Adhesion Molecule-1 (sICAM-1), soluble Endoglin (sEndoglin), C-reactive protein (CRP), Chitinase-3-Like Protein-1 (CHI3L1), and complement component C5a (C5a) using commercially available enzyme linked immunosorbent assay (ELISA) kits (Duosets, R&D Systems, Minneapolis, MN). All laboratory procedures followed the manufacturers’ protocol (R&D System Duosets) with two notable exceptions; our laboratory used a longer incubation period (overnight at 4°C) and samples were developed using ExtrAvidin (1:1000 dilution, Sigma-Aldrich Co.) and SIGMAFAST p-Nitrophenyl phosphate Tablets (Sigma-Aldrich Co.). Sample dilutions were determined based on previous pilot studies and results in our laboratory. Analysis was performed blinded to the patient group. Biomarkers were selected based on previous studies implicating these factors, or the pathways they reflect, as potential mediators of adverse pregnancy outcomes.

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Manufacturer protocol

Download the product protocol from R&D Systems for Human Angiopoietin-like 3 DuoSet ELISA below.

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