O phenylenediamine dihydrochloride substrate

Secreted-human NM23 was detected by ELISA using an NM23 isozyme non-specific antibody that recognizes human NM23. Ninety-six well plates (Corning, NY) were coated with recombinant NM23 protein as standard sample (Abnova, Taiwan), or serum test proteins (experimental). Standard Nm23 samples were employed over a range from 0.03 to 100 ng/mL in phosphate buffered saline (containing 0.05% sodium azide); 50 μl aliquots were added to wells and the plate incubated for 2 hr at room temperature (RT) and then overnight at 4°C on a slow shaker. Blocking buffer (100 μl) containing 5% BSA and Tween-20 (0.05%) in phosphate-buffered saline (PBST 0.05%) was added and incubated for 1.5 hr at RT. Plates were then washed once with 150 μl of PBST, incubated with primary mouse anti-NM23 antibody (Abcam, Cambridge, MA USA) diluted with PBST buffer containing 1 mM EDTA and 0.25% BSA (100 μl) for 2 hr at 37°C with slow rocking, and then washed 3 times with 150 μl PBST. Plates were incubated with rabbit anti-mouse IgG horseradish peroxidase (HRP)-conjugate (Southern Biotech, Birmingham, AL) for 2 hr at 37°C with slow rocking and washed 3 times with PBST. Plates were developed by addition of 100 μl of o-phenylenediamine dihydrochloride substrate, (Sigma St. Louis, MO) and absorbance measured at 490 nm using a microplate reader.