Ziva® Ultra SEAP Plus
Ziva® Ultra SEAP Plus is by far the most sensitive SEAP detection assay available today. The Ziva Ultra SEAP Plus Kit assay’s Signal-to-Noise ratio is ~100X or more, sensitive in detecting both Heat Stable and Non-Heat Stable APs than the next most sensitive chemiluminescent SEAP assay kit from Competitor X, and far more sensitive than either fluorescent or colorimetric SEAP detection kits. Competitor X’s SEAP assay was chosen for product performance comparisons to the Ziva Ultra SEAP Plus assay because Competitor X’s marketing claims that their SEAP assay provides higher signal intensity, higher signal/noise ratios (~ 10 X higher) and lower-end sensitivity compared to other previous chemiluminescent based SEAP assays (this claim excluded Ziva).
Figure 1. A HPAP master stock solution was prepared by obtaining Human Placental Alkaline Phosphatase (HPAP) obtained from Competitor X commercial SEAP Assay Kit, and spiking the HPAP into DMEM cell culture medium plus 10% FBS. Test dilutions were prepared by serial diluting the HPAP master stock solution in DMEM plus 10% FBS. Five microliters (5 µL) of each test dilution were assayed in triplicate, using a white 96-well microtiter plate according to the instructions in each respective package insert including the heat inactivation step. Chemilumunescence was measured using Jaden’s Insight-Mi® microplate luminometer.
The Ziva Ultra SEAP Plus assay is a rapid, simple, easy-to-use chemiluminescent assay detection system designed for the ultrasensitive detection of the SEAP reporter gene AP product, and other Heat Stable (HS) or Non-Heat Stable (NHS) AP. Transfection is a laboratory method that deliberately introduces nucleic acids into living eukaryotic cells. The SEAP reporter gene is one of the most common genes chosen to be coupled with a gene expression promoter and a protein gene for introduction into cells by transfection. The SEAP reporter protein molecule is a recombinant truncated form of Human Placental AP (PLAP) that does not possess the capacity to bind to the cell membrane and is secreted by the cell into the biological fluid the cells reside in. The presence of SEAP activity in either the cells or culture media is used to monitor the effectiveness of the transfection and the expression of the SEAP attached protein in the cells. It is now possible to detect PLAP beginning 2 to 3 hours after the start of transfection, because Ziva Ultra SEAP Plus can detect sub-femtogram levels of PLAP. The detection of the SEAP reporter gene expression product PLAP can also be used for in vivo studies.
Alternatively, full length PLAP vectors are designed so that the PLAP produced by the transfected cell binds to the cell membrane. This type of vector is often used for in vivo marking of cells. Heat stable APs such as PLAP and Germ Cell Alkaline Phosphatase (GCAP) are expressed in some cancer tissues (lung, ovary, uterus, testis, chorion, Hela, gastrointestinal) but not in the normal tissue the cancer arises from. Ziva Ultra SEAP Plus can also be used to monitor stem cell AP expression that marks differentiation. Chemiluminescent assays are by far the most sensitive AP detection assays available.
Ziva Ultra SEAP plus was compared to Competitor X”s SEAP assay’s detection of the presence of non-heat stable alkaline phosphatase (AP). Table 1 presents the Signal to Noise (SN) ratio obtained when equal amounts of the AP of interest are compared.
| Non-Heat Stable Mammalian Alkaline Phosphatase | Assay Comparison of Signal-to-Noise (SN) (SN Ziva / SN Competitor X) |
|---|---|
| Calf Intestine | ≥ 100 |
| Human Bone | ≥ 100 |
| Human Liver | ≥ 100 |
Table 1. Non-Heat Stable Alkaline Phosphatase (AP) test samples were prepared by spiking AP into DMEM cell culture medium plus 10% FBS. Two microliters (2 µL) of each test sample were assayed in triplicate, in a white 96-well microtiter plate according to the instructions in each respective package insert. Chemilumunescence was measured using Jaden’s Insight-Mi® microplate luminometer.
Ziva Ultra SEAP Plus Assay Advantages:
- The capability of detecting at a 2/1 S/N ratio as few as ~10e-21 moles (~500 to 1000 molecules) of Heat stable human PLAP or non-Heat Stable calf intestine AP.
- An assay linear dynamic range of 10e5 where the linear signal detection range capability of the luminometer used to read the signal allows it.
- Transfection efficiency kinetic measurements of SEAP gene expression in cell culture media is detectable as early as 2-4 hours after the start of transfection, compared to much later ( ~20 to 30 hours) when using competitor’s SEAP assays.
- Biomarker detection: because no chemical AP inhibitors are used in the assay both HS (PLAP, GCAP among others) and NHS AP (bone, serum, liver, kidney, intestine) can be effectively and ultrasensitively detected by the assay.
- Multiple liquid sample matrices can be assayed: cell culture media, serum, urine, and other biological fluids, saving cells for other applications.
- Assay cells containing AP directly, which is useful in monitoring stem cell differentiation or other research applications.
- A persistent and stable signal detection glow that provides great flexibility and allows measurement of the assay signal over a wide time intervals (half time of decay of the glow chemiluminescent signal is =20 hrs at room temperature).
- The assay is simple to perform and rapid (~ 1 hour) and requires only the sample and the SSPS and Substrate reagents provided by the kit.
- The assay uses very small test sample volumes (1-10 microliters).
- All necessary reagents are contained in the kit.