Bioassays for Cytotoxicity Applications -

Applications & Products

KILR^® Cytotoxicity Bioassays

Robust, Ready-to-Use Bioassays to Measure Direct Cell Death

The KILR cytotoxicity assay platform provides a robust and sensitive solution to measure the killing of antigen-expressing target cells in co-culture with effector cells. The platform allows for the quantification of direct cell death through multiple mechanism of actions (MOAs), including ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), CDC (complement-dependent cytotoxicity, and T-cell redirection.

Eurofins DiscoverX^®’s KILR bioassays are plate-based, homogeneous ready-to-use (RTU) cell-based assay kits that include all necessary reagents to run the assay. These physiologically relevant, robust bioassays are fit-for-purpose for use in screening and characterization applications to potency testing in quality control (QC) lot-release programs. The KILR RTU bioassays demonstrate extensive lot-to-lot consistency in assay performance and help ensure long-term assay reproducibility with high signal-to-background ratios, key factors in potency lot-release testing programs. The KILR platform provides the flexibility to use your effector cells of choice. Additional custom assay development capabilities are available to generate optimized bioassays with your target cell model of interest and for relevant data generation.

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Highlights of KILR Cytotoxicity Bioassays

Multiple Applications – Analyze ADCC and ADCP in screening and characterization applications to potency testing in QC lot-release programs
Comparable Performance – Excellent concordance in assay performance between continuous culture and RTU bioassay formats
Simple Protocol, Fast Results – Easy-to-run, rapid homogeneous protocol amenable to implementation in multiple labs and high-throughput format for increased efficiency
Reproducible Results – Ensure long-term assay reproducibility with high signal-to-background ratios

High Repeatability and Reproducibility of Results

KILR ADCC bioassays deliver high repeatability and reproducibility. A. A dose response of the anti-CD20 antibody Rituximab was evaluated in the respective target cells by a single analyst on three different days using KILR Raji bioassay target cells and KILR CD16 Effector Cells. The results demonstrate the low variability in EC50, S/B (signal-to-background), and EMAX values over the three days indicating the high repeatability of the ADCC assay. Comparable data was obtained with the KILR Daudi model (data not shown). B. Functional response analysis of KILR ADCC bioassays by two analysts over two days shows excellent concordance in curve shape, S/B, and EC₅₀.

Higher Percentage of Phagocytosis Seen with Plate-based KILR ADCP Assay Compared to Flow-based

Higher Percentage of Phagocytosis Seen with the Plate-based KILR ADCP Assay Compared to the Flow-Based Assay Format

Comparison of rituximab-mediated ADCP in two different assay formats – flow cytometry vs the KILR enzyme fragment complementation (EFC) format. Monocytes from the same healthy donor were isolated and differentiated to M0 macrophages prior to use in each assay. A. Expression of CD11b and CD14 was assessed on the polarized macrophages by flow cytometry at the time of harvest to confirm the differentiation state. B. Flow-based and C. KILR EFC-based assay results normalized to vehicle-treated cells to calculate % ADCP. Overall, a higher percentage of phagocytosis was observed when using the KILR assay, indicating a more effective ADCP assay format. E:T = Effector cells (macrophage):target.

Well-Structured Development Process Delivers Robust and Reproducible Assays

A well-structured process for bioassay development yields assays that are robust and reproducible. The process begins with selecting the assay that most appropriately reflects the MOA of the drug, followed by optimization of assay parameters. Optimization through qualification steps includes refining conditions with the originator drug or reference molecule to establish robustness, precision, accuracy, and linearity of assay.

Key Resources

KILR Bioassay Kits

Bioassay Kit Name	Cancer Cell Type	Target	Assay Measures	Drug Name	Bioassay Kits & Configuration
KILR Raji ADCC Bioassay Kit	Burkitt’s lymphoma	CD19, CD20, CD38	Antibody-Dependent Cellular-Cytotoxicity	Rituximab	97-1012Y026-00169 (2-plate) 97-1012Y026-00170 (10-plate)
KILR Daudi ADCC Bioassay Kit	Burkitt’s lymphoma	CD19; CD20; CD38	Antibody-Dependent Cellular-Cytotoxicity	Rituximab	97-1009Y025-00171 (2-plate) 97-1009Y025-00172 (10-plate)
KILR Raji ADCP Bioassay Kit	Burkitt’s lymphoma	CD19; CD20; CD38	Antibody-Dependent Cellular-Phagocytosis	Rituximab	97-1012Y026-00179 (2-plate) 97-1012Y026-00180 (10-plate)
KILR Daudi ADCP Bioassay Kit	Burkitt’s lymphoma	CD19; CD20; CD38	Antibody-Dependent Cellular-Phagocytosis	Rituximab	97-1009Y025-00177 (2-plate) 97-1009Y025-00178 (10-plate)

Request qualification data set for KILR^® Raji ADCC Bioassay Kit using rituximab.

The KILR platform is based on the industry-validated Enzyme-Fragment Complementation (EFC) technology. EFC is based on two recombinant β-galactosidase (β-gal) enzyme fragments that act as an enzyme acceptor (EA) and an enzyme donor (ED). Separately, the fragments are inactive, but when combined, they form an active β-gal enzyme that hydrolyzes its substrate to produce a chemiluminescence signal.

KILR ADCC Assay Principle

The KILR ADCC assay principle. Target cells expressing the relevant antigen are engineered to stably express a housekeeping protein tagged with a reporter β-gal ED fragment called enhanced ProLabel^® (ePL). The β-gal enzyme is inactive when the reporter fragment (also called the KILR reporter protein) is not paired with its larger β-gal EA fragment. On incubation of KILR target cells with a test antibody and appropriate effector cells (e.g. KILR CD16 Effector Cells), effector cell-mediated killing releases the tagged protein into the media. The presence of both EA and ED fragments leads to the formation of the active β-gal enzyme that hydrolyzes the substrate to give a chemiluminescent output detected on any benchtop luminometer.

KILR ADCP Assay Principle

The KILR ADCP assay principle. ADCP measures antibody-dependent phagocytosis of target cells, typically mediated by primary human macrophages. KILR target cells, containing the KILR reporter protein (including the β-gal ED fragment also called ePL), undergo phagocytosis by effector cells (e.g. macrophages differentiated from monocytes) leading to lysosomal degradation of the KILR reporter protein. In this assay, the KILR target cells are opsonized with the antibody of interest, then co-cultured with the effector cells. Macrophages perform phagocytosis of the target cells. All non-phagocytosed cells are lysed and the detection reagent containing the complementing β-gal EA fragment is added to the lysed cells. Complementation of ED and EA with the addition of substrate results in the detection of the non-phagocytosed KILR protein present. A high chemiluminescent signal indicates minimum killing, while a low signal correlates to higher phagocytosis or increased ADCP activity. Note: other immune effector cells can also be used for the ADCP assay.

Perform Potency Assays

Qualification data for KILR Raji Bioassay using rituximab demonstrating the assays ability to determine relative potencies. A. Study design for qualification of KILR Raji ADCC Bioassay. Five nominal concentrations of rituximab were evaluated over a range of 50% to 150%. Repeatability (4 runs) was assessed at the 100% nominal concentration by a single analyst. Intermediate precision incorporated several sources of variability, including multiple analysts, multiple days, 2 lots of KILR Raji Bioassay target cells, and 3 different lot of KILR CD16 Effectors. B. Results obtained from the qualification study indicate very good accuracy, high repeatability, intermediate precision, and dilutional linearity for KILR Raji Bioassay.

KILR® Cytotoxicity Bioassays