Secreted alkaline phosphatase (SEAP) is widely used as a reporter to study gene expression. SEAP is secreted into the culture supernatant of the transfected cell lines. SEAP activity in the culture medium reflects changes in intracellular concentrations of SEAP mRNA and protein. The changes of gene expression can be easily assayed using the same cultures by repeated sampling. Cell Technology’s aCellaTM – SEAP detection kit utilizes a Chemiluminescent-based assay technique to detect SEAP reporter gene expression. It provides an extremely sensitive tool to quantitate SEAP and analyze transcriptional and promoter activity in transfected cells.

In the assay, a chemiluminescent substrate was used to detect the Secreted Alkaline Phosphatase in cell culture. The stabilized 1, 2-dioxetane substrate provide high signal to noise ratio, wide dynamic range, rapid results and excellent reproducibility. The SEAP enzyme transfers the phosphoryl residue via a phosphoryl-enzyme intermediate. The intermediate is not stable and decomposed and emits light for detection. The assay can detect alkaline phosphatase at attogram level. The kit provides sample material for 100 assays in a 96-well plate format.

Figure 1. Standard curve of Alkaline Phosphatase (AP). 50 µl serial dilutions of AP (Starting dose 100 mU in tubes) were added to the wells of 96-well luminescent plate. 50 µl of Chemiluminescent substrate was added and the plate was read immediately with a luminometer.

Figure 2. Detection of the functional activity of NF-ĸB/SEAP stable reporter cell line. NF-ĸB/SEAP reporter cells (IML-101) were plated in 96-well plates at 50,000 cells/well. After 24 hours of incubation, cells were stimulated with different doses of TNFα for another 24 hours. 50 µl culture supernatant was added to the wells of 96-well white opaque luminescent plate. 50 µl of chemiluminescent substrate was added and the plate was read immediately with a luminometer.

Figure 3. Inhibition of TNFα activity by a quinazoline TNFα inhibitor (EMD Millipore Cat# 545380-34-5) with NF-ĸB/SEAP stable reporter cell line. NF-ĸB/SEAP reporter cells (IML-101) were plated in 96-well plates at 50,000 cells/well. After 24 hours, 10 ng/ml of TNFα and different dose of TNFα inhibitors were added into each wells. Cells were incubated at 37°C for 24 hours. 50 µl culture supernatant was added to the wells of 96-well white opaque luminescent plate. Then 50 µl of Chemiluminescent substrate was added and the plate was read immediately with a luminometer.