ELISA protocol

The mouse and rat endostatin ELISA (BI-20742MR, Biomedica, Vienna, Austria) is based on a sandwich type format, where the affinity purified polyclonal goat antibody is immobilized on a 96-well microtiter plate. The detection antibody is an affinity purified polyclonal goat antibody conjugated to horseradish peroxidase and diluted in a peroxidase-stabilizing buffer system.

The assay includes seven standards (0 (STD1), 1 (STD2), 2 (STD3), 4 (STD4), 8 (STD5), 16 (STD6) and 32 (STD7) nmol/L) and one control, which contain recombinant mouse endostatin diluted in a protein-based buffer system. Standards, control and samples are pre-diluted 1+50 in a tube using the protein-based assay buffer. 50 μl of these-predilutions are pipetted into the pre-coated plate and subsequently 50 μl of detection antibody solution is added per well. The plate is covered with an adhesive strip and incubated for two hours at room temperature. Each well is aspirated and washed five times before adding 100 μl of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solution to each well. After 30 minutes of incubation at room temperature 50 μl of the stop solution is added and the optical density (OD) is obtained with a spectrophotometer using 450 nm as reference and 630 nm as correction wavelength (BioTec, Winooski, VT, USA). The standard curve is fitted using the 4PL algorithm. Concentration of samples and controls are calculated based on the standard curve. Our assay has been commercialized by Biomedica (BI-20742MR, Biomedica, Vienna, Austria).