Advantages Of The KILR Assay
- Unparalleled Specificity – Signal only from dead target cells
- Easy to use – Simple add and read protocol with chemiluminescent readout
- Exquisite Sensitivity – Detect as few as 75 dead cells with high reproducibility
- Ultimate Flexibility – Ability to run cytotoxicity assay from 30 minutes to 72 hours
Highly Specific – KILR detection protein only present in target cells
Target cells expressing the receptor antigen of choice can be engineered to stably express a protein tagged with enhanced ProLabel (ePL), a β-gal reporter fragment, using the KILR Retroparticles. When the stable target cell line is used in a cytotoxicity assay, and its membrane is compromised due to cell death, it will release the tagged protein into the media. We can detect this KILR reporter protein in the media by the addition of detection reagents containing the enzyme acceptor (EA) fragment of the β-gal reporter. This leads to the formation of the active β-gal enzyme which hydrolyzes the substrate to give a chemiluminescent output, detected on any bench top luminometer.
Easy to use – Simple, one-step add and read protocol
A simple non-radioactive, add and read protocol with a chemiluminescent output that can be read on any benchtop luminometer. Using the KILR assays, you can eliminate the need to load target cells prior to every experiment, the use of radioactivity, and reduce the number of steps, increasing the efficiency of the lab.
Highly Sensitive – Robust detection of molecules with low-level toxicity
KILR H322 cells were plated at various densities with primary human PBMCs and Cetuximab, an anti-EGFR drug for colorectal cancers, to measure ADCC response. ADCC is a known mechanism of action for Cetuximab, where the antibody activates immune cells to kill target cancer cells. Target cell death is reported as % Lysis, a ratio of experimental signal to total signal generated when the cells are lysed with detergent. The data on the left shows we can robustly detect 3% Lysis in the KILR H322 cells when a total of 2500 KILR cells are plated in the well, indicating that we have detected the death of 75 cells inside the well. This exquisite sensitivity of the assay demonstrates its value in applications such as screening and lead optimization, where sensitivity is critical to identify and optimize lead drug candidates.
Target cells expressing the antigen of interest are engineered to stably express a housekeeping gene that is tagged with ProLabel® (ePL), a β-gal reporter fragment in our proprietary EFC technology (What is EFC?
). The KILR Detection Reagents contains the other β-gal fragment, EA, which naturally complements with the ePL fragment to create the active β-gal protein ( Figure A) that then hydrolyzes substrate to generate chemiluminescence. The target cells are stably engineered to express the KILR reporter protein. In Figure B, the well on the left contains healthy, intact target cells that are alive in the presence of immune effector cells. When KILR Detection Reagent is added, we cannot detect chemiluminescence as the KILR reporter protein does not leak out into the media through the intact cell membrane of the live cells. Alternatively, in the well on the right, the target KILR cells are killed by the immune effector cells, releasing the KILR reporter protein into the media. Addition of the KILR Detection Reagent leads to the recognition of this reporter protein in the media and the generation of a chemiluminescent signal that is proportional to the number of dead cells. Death of any other cell type, including immune effector cells present within the co-culture will not affect the assay output, giving the KILR assay unparalleled specificity to detect target cell death within a co-culture assay.
Example of an ADCC assay with KILR cell lines
A. Data demonstrating Trastuzumab (Herceptin®)-mediated cytotoxicity in SKBR3 cells. Cells were opsonized with a dose response of Herceptin (anti- ErbB2/Her2) and incubated with NK cells. This demonstrates that the assay is able to robustly detect ADCC with Transtuzumab, an antibody known to cause an ADCC response.
B. Data demonstrating rituximab-mediated ADCC in CD20+ ARH-77 target cells opsonized with a dose response of rituximab (anti- CD20) incubated with NK cells. EC50 of 3.6 ng/mL is consistent with EC50 observed using other ADCC assay formats (e.g. chromium-51 release; europium) indicating similar pharmacology and ability to detect ADCC, but in a much simpler way.