Human gm csf elisa kit

Human cytokine antibody array (ab133998 from Abcam) was used in accordance with the manufacturer's instructions. Briefly, the membranes containing cytokine antibodies were blocked, incubated with 1 mL conditioned medium (CM) for 2 h at room temperature, washed, and then incubated with biotin‐conjugated antibodies for 2 h and with horseradish peroxidase‐linked secondary antibody for another 2 h. The membranes were incubated with chemiluminescent substrate. The ChemiDoc XRS system (BioRad, Hercules, CA, USA) was used to detect the chemiluminescence. For quantitation of GM‐CSF, the Human GM‐CSF ELISA Kit (ab100529 from Abcam) was used in accordance with the manufacturer's instructions. In brief, GM‐CSF standard and samples were pipetted into the wells containing human GM‐CSF‐specific antibody and incubated at room temperature for 3 h. The wells were washed and then biotinylated human GM‐CSF antibody was added, followed by incubation for 45 min. After removing the unbound biotinylated antibody by washing, horseradish peroxidase‐conjugated streptavidin was added. The wells were again washed, and TMB substrate solution was pipetted into the wells and incubated for 30 min, followed by addition of a stop solution. The intensity of the color was measured at 450 nm.