Ma5 14732

We used recombinant human p-selectin/CD62P (ADP3-050, R&D systems), recombinant human apolipoproteinA1 (3664-AP, R&D systems) or blood proteins harvested by centrifugation or three layered filter system to evaluate the immunoassay using color dye. To measure the limits of detection of the assay, we used serial diluted recombinant proteins. One ul of recombinant protein solution was applied to the filter system. Filter three was blocked with 1% bovine serum albumin (A7906, Sigma) in phosphate buffered saline for 10 minutes at room temperature. After brief washing with 0.1 M Tris buffer solution for 5 minutes, primary antibodies (anti-human SELP/P-selectin/CD62P antibody (LS-C296359, LSBio) and mouse anti-human ApoA1 antibody (MA5-14732, Invitrogen) were used for antigen-antibody reaction. HRP-linked secondary antibody (#.7074, Cell Signaling) was incubated for one hour at room temperature and followed by color reaction with 3,3´,5,5´-tetramethylbenzidine (TMB)-D solution (4600, Kem-En-Tec Diagnostics). When HRP conjugated mouse anti-human IgG antibody (SA1-35470, ThermoFisher Scientific) and HRP linked goat anti-human IgM antibody (31415, ThermoFisher Scientific) were used, color reaction with TMB solution was made directly. Enhanced chemiluminescence (ECL) using PicoEPD Western reagent (EBP1073, ELPIS-BIOTECH) was performed to compare the result with TMB solution.

To confirm the presence and size of target proteins we performed sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot assay. After incubation with primary antibodies (human SELP/P-selectin/CD62P antibody (LS-C296359, LSBio), human IgG antibody (SA1-35470, ThermoFisher Scientific), human IgM antibody (31415, ThermoFisher Scientific), human apolipoproteinA1 antibody (MA5-14732, Invitrogen) for 1 hour at room temperature, horseradish peroxidase (HRP)-linked secondary antibody (#.7074, Cell Signaling) was added for one hour at room temperature. Enhanced chemiluminescence (ECL) reaction was done to visualize the protein band using PicoEPD Western reagent (#.EBP1073, ELPIS-BIOTECH).