The HTRF Human Androgen Receptor Variant 7 assay quantifies the expression level of Androgen Receptor in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Human Androgen Receptor Variant 7 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of Androgen Receptor in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Human Androgen Receptor Variant 7 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Detection of Human Androgen Receptor Variant 7 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

HEK293 cells were plated in a T175 flask in a complete culture medium for 24h at 37°C, 5% CO2, then were transfected with Androgen Receptor Full Length (AR FL) or Androgen Receptor Variant 7 (AR-V7) plasmids. After 24h, the cells were lysed with 3 mL of lysis buffer# 3 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using lysis buffer# 3 (1X) , then 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Androgen Receptor Variant 7 (AR-V7) reagents were added. The HTRF signal was recorded after a 2h incubation at room temperature.

As the HEK293 cells did not express Androgen Receptor or Androgen Receptor Variant 7, the non transfected cells did not give any signal. Similarly, AR FL transfected cells were not detected with the AR-V7 assay. The AR-V7 transfected cells gave a very high HTRF signal, demonstrating the specificity of the assay.

Human breast cancer cells MDA-MB-451 and prostate cancer cells LnCap and 22RV1 were seeded at 100,000 cells / well in a 96-well microplate. After a 24h incubation, the cells were lysed with supplemented lysis buffer #3 (1x).

16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Androgen receptor Full Length (ARFL), HTRF Androgen Receptor Variant 7 (AR-V7), or HTRF Total Androgen Receptor reagents. The HTRF signal was recorded after an overnight incubation.

As expected, little Variant 7 (AR-V7) was detected in the MDA-MB-451 and LnCap cell lines, while it was well detected in the 22RV1 cell line.

The Total Androgen Receptor assay able to detect Full Length, as well as all variants, giving a high signal in the 3 cell lines.

Full Length Androgen Receptor was well detected in the 3 cell lines.

22RV1 prostate cancer cells were cultured in a T175 flask in a complete culture medium for 24h at 37°C, 5% CO2. The cells were then lysed with 3 mL of supplemented lysis buffer #3 (1x) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer #3 (1x), and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Androgen Receptor Variant 7 (AR-V7) detection reagents.

Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF Androgen Receptor Variant 7 (AR-V7) assay was 32 times more sensitive than the Western Blot technique.

Androgen Receptor (AR) is a type of nuclear receptor that is activated by androgen hormones, like testosterone (T) and dihydrotestosterone (DHT). The androgen hormones cross the cell membrane to bind directly onto the Androgen Receptor, inducing homodimerization of the Androgen Receptor and permiting nucleus translocation. The Androgen Receptor dimers then bind to the Androgen Response Element (ARE) to lead gene expression, resulting in cell division, proliferation, differentiation, apoptosis, and angiogenesis.

The Androgen Receptor Variant 7 (AR-V7), a splice variant of the androgen receptor mRNA resulting in the truncation of the ligand-binding domain, is constitutively active and widely expressed in human cancer cell lines. In the same way as the Full Length, ARV7 dimerize  to translocate into the nucleus to activate the same effectors.

To be added to total androgen receptor kit as well.