Description
Trophoblast Cell Surface Antigen 2 (TROP2) is a transmembrane glycoprotein that regulates cell proliferation, survival, and invasion through both cell surface signaling and intracellular domain-mediated transcriptional activation. TROP2 undergoes regulated intramembrane proteolysis, releasing its intracellular domain to translocate to the nucleus and activate target genes involved in cell cycle progression and survival. It is normally expressed in trophoblasts and epithelial tissues but becomes overexpressed in numerous solid tumors including breast, lung, colorectal, and pancreatic cancers. TROP2 overexpression correlates with poor prognosis, increased metastasis, and chemotherapy resistance. Its selective expression in tumors versus normal tissues makes TROP2 an attractive target for antibody-drug conjugates (ADCs) and immunotherapies, with several TROP2-targeted therapeutics showing clinical efficacy in advanced cancers.
The AlphaLISA SureFire Ultra Human Total TROP2 is a sandwich immunoassay for the quantitative detection of total TROP2 in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
Total-AlphaLISA SureFire Ultra two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Total-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.
Selectivity of TROP2 Total assay
TROP2 levels were assessed in MCF7 wild type (WT) and TROP2 KO (Abcam, ab286330) cell lines cultured to confluency in T175 flasks at 37°C, 5% CO2.
Each flask was lysed in 20 mL of Lysis Buffer for 10 minutes at RT with shaking. Lysates were serially diluted in Lysis Buffer and TROP2 levels were evaluated using the AlphaLISA SureFire Ultra assay kit. For the detection step, 10 µL of cell lysate was transferred into a 384-well white OptiPlate, followed by 5 µL Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
TROP2 was detected in WT but not in the KO cells. This demonstrates the selectivity of the assay for the detection of TROP2 protein.
Expression of TROP2 in various cell lines
Adherent cells were seeded at 4,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2. Cells were lysed with 200 µL of Lysis Buffer. Suspension cells were washed with HBSS and lysed with Lysis Buffer at 2 x 106 cells/mL.
TROP2 expression was evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 200 adherent cells or 20,000 suspension cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, TROP2 is highly expressed in A431, RT4 and MCF7 cell lines while no signal was detected in A549, HEK293, THP-1 and RPMI 8226 cells.
