Mouse C-Reactive Protein/CRP 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

Left ventricular myocardial concentration of GAL-3, c-reactive protein (CRP), neutrophil elastase, myeloperoxidase (MPO), interleukin-6 (IL-6), and cleaved caspase-3 were determined using DuoSet enzyme linked immunosorbent assay (ELISA) development kits [mouse galectin-3 (DY1197), mouse CRP (DY1829), mouse neutrophil elastase (DY4517), mouse MPO (DY 3667), mouse IL-6 (DY 406) and mouse cleaved caspase-3 (DYC 835), R&D Systems, Minneapolis, MN, USA] for sandwich ELISA, using standard procedure according to the manufacturer’s instructions. The levels were normalized to total protein concentrations. Briefly, 96-well plates Nunc-Immuno Plate MaxiSorp Surface (NUNC Brand Products, A/S, Roskilde, Denmark), were coated with antibody specific our proteins of interest. Biotinylated detection antibody and streptavidin conjugated horseradish peroxidase were used for detection of captured antigens. The plates between steps were aspirated and washed 3 times using ELISA plate washer (BioTek ELx50). Captured proteins were visualized using tetramethylbenzidine (TMB)/hydrogen peroxide. Absorbance readings were made at 450 nm, using a 96-well plate spectrophotometer (BioTek ELx800). Concentrations in the samples were determined by interpolation from a standard curve. Standards and samples were assayed in duplicate.

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Manufacturer protocol

Download the product protocol from R&D Systems for Mouse C-Reactive Protein/CRP DuoSet ELISA below.

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