This assay requires a six-hour incubation of cells with the antibody of interest for optimal performance, typically in a standard cell culture incubator. This paper evaluates an alternative method of performing the assay, using Tecan’s Infinite 200 PRO and Gas Control Module (GCM) (figure 1). This multimode reader set-up offers rigorous control of the temperature and CO2 and O2 partial pressures within the measurement chamber, allowing in-reader incubation and complete automation of the assay.
Target cells (Raji cells and WIL2-S cells) were thawed and mixed with assay buffer (RPMI 1640 medium + 4 % low IgG serum), then plated into the inner 60 wells of two 96-well plates (perimeter wells filled with assay buffer). A dilution series of control antibody, Anti-CD20, was added to both plates in final concentrations from 6 μg/ml to 0.73 ng/ml for Raji cells, and 4 μg/ml to 0.14 ng/ml for WIL2S cells. Wells without antibody served as negative controls, and wells containing only buffer served as reagent background controls.
Effector cells were prepared in the same manner as the target cells, and added to the inner 60 wells of both plates, then briefly mixed on an orbital shaker. The plates were then incubated in the Infinite 200 PRO or a tissue culture incubator at 37 °C, 5 % CO2 for six hours.
Following incubation, BioGlo™ reagent (Bio-Glo Luciferase Assay Substrate in Bio-Glo Luciferase Assay Buffer) was added to the experimental and background control wells, and incubated within the Infinite 200 PRO measurement chamber at 37 °C for a further five minutes prior to luminescence measurements.
The results of the ADCC Reporter Bioassay with Raji or WIL2-S target cells were comparable in both fold induction and potency whether the incubation was performed in an Infinite 200 PRO with GCM or in a standard tissue culture incubator (figure 2). The potencies obtained were 66 ng/ml and 78 ng/ml respectively for Raji cells, and 36 ng/ml and 37 ng/ml respectively for WIL2-S cells.
The Infinite 200 PRO with GCM is functionally equivalent to a tissue culture incubator for the reporter induction phase of the ADCC Reporter Bioassay. This study showed that the convenience of using a kit containing frozen, thaw-and-use cells was further enhanced by the use of a reader that enabled both temperature and gas control, effectively mimicking the conditions provided by a standard cell culture incubator.
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