U-bottom microplates (untreated) were coated overnight with antibody and fragments, fibronectin or BSA with 100 mL of 10 μg/mL protein in 0.2 M bicarbonate buffer, pH 9.4 at 4°C. Wells were washed and blocked with 200 mL PBS/1% BSA for 1 hr at room temperature. 2.5×104 Jurkat or CHO cell transfectants expressing CD16a, 32a or 64a were added with or without sCA125 to wells in a total volume of 100 μL ADCC assay buffer (Promega). Microplates were placed at 37°C/5% CO2 for 5 hr then imaged using an EVOS imaging system (Thermo-Fisher, Waltham, MA). Cell cluster diameter was quantitated by A405 using a SpectraMax M5 (Molecular Devices, Downingtown, PA). sCA125 competition studies were done using 50 μg/mL of antibody or antibody fragment. sCA125-mesothelin absorption is described in Supplementary Methods .
Adcc assay buffer
Manufactured by Promega
The ADCC assay buffer is a reagent used in the performance of antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The buffer provides the necessary components to facilitate the interaction between effector cells and target cells, which is a key step in the ADCC process. The specific composition and formulation of the buffer are designed to support the assay conditions required for the evaluation of ADCC activity.
Automatically generated - may contain errors
Lab products found in correlation
96 well cell culture plate, by Greiner (2 mentions)
Bio glo luciferase assay reagent, by Promega (2 mentions)
Adcc bioassay effector cells, by Promega (2 mentions)
Spectramax l, by Molecular Devices (2 mentions)
Synergy h1, by Agilent Technologies (2 mentions)
Bioglo luciferase assay reagent, by Agilent Technologies (2 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Evos imaging system, by Thermo Fisher Scientific (1 mentions)
5 protocols using adcc assay buffer
1
Antibody-Dependent Cell-Mediated Cytotoxicity Assay
2
ADCC Reporter Bioassay for Antibody Screening
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Example 9
ADCC Reporter Bioassay
2.5×104 HCC1428 cells in 100 μL culture medium/well were incubated in 96-well cell culture plate (Greiner, Cat #655083-1) at 37° C. in a humidified 5% CO2 incubator (NUAIRE, Cat #NU-4750) overnight. Culture medium was replaced with 2× serial dilution of 37.5 μL OBI-898 from 20 to 0.000305 μm/mL in ADCC Assay Buffer (Promega, Cat #G708A, G711A). In addition, background wells which contained no antibody were included. In each well, 37.5 μL of 7.5×104 ADCC Bioassay Effector cells (Promega, Cat #G701A) were added. Induction was performed for six hours and 75 μL of Bio-Glo Luciferase Assay Reagent (Promega, Cat #G719A, G720A) was added. After 15 minutes, luminescence (RLU, relative light unit) was determined using a microplate luminometer, SpectraMax L (Molecular Devices, Sunnyvale, Calif.).
Luminescence induction of fold changes was calculated by the ratio of relative light unit (RLU) (induced) to RLU (no antibody control). EC50 was determined by plotting x (concentration in μg/mL)−y (the induction of fold change) and fitting the data in a 4PL nonlinear regression model by PRISM 6 Software.
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3
ADCC Bioassay for Antibody Characterization
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Monoclonal antibodies were serially diluted in ADCC assay buffer (Promega). Target cells (A549-H1 HA, A/California/04/2009) were added in a white flat-bottom 96-well plate at 1.25 × 104 cells per well in 25 μl, then serially diluted antibodies were added to each well (25 μl per well), and the antibody and cell mixture was incubated for 10 min at room temperature. Effector cells (Jurkat-FcγRIIIa) for the ADCC Bioassay are thawed and added at a cell density of 7.5 × 104 per well in 25 μl (effector to target ratio of 6:1). Plates were incubated for 20 h at 37 °C with 5% CO2. Activation of human FcγRIIIa (V158 or F158 variants) in this bioassay results in the NFAT-mediated expression of the luciferase reporter gene. Luminescence is therefore measured with a luminometer (Synergy H1, Biotek) using the BioGlo Luciferase Assay Reagent according to the manufacturer’s instructions. The data (that is, specific FcγRIIIa activation) are expressed as the average of relative luminescence units (RLU) over the background by applying the following formula: (RLU at concentration x of monoclonal antibodies − RLU of background).
4
ADCC Reporter Bioassay for Antibody Characterization
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Example 9
ADCC Reporter Bioassay
2.5×104 HCC1428 cells in 100 μL culture medium/well were incubated in 96-well cell culture plate (Greiner, Cat #655083-1) at 37° C. in a humidified 5% CO2 incubator (NUAIRE, Cat #NU-4750) overnight. Culture medium was replaced with 2× serial dilution of 37.5 μL OBI-898 from 20 to 0.000305 μg/mL in ADCC Assay Buffer (Promega, Cat #G708A, G711A). In addition, background wells which contained no antibody were included. In each well, 37.5 μL of 7.5×104 ADCC Bioassay Effector cells (Promega, Cat #G701A) were added. Induction was performed for six hours and 75 μL of Bio-Glo Luciferase Assay Reagent (Promega, Cat #G719A, G720A) was added. After 15 minutes, luminescence (RLU, relative light unit) was determined using a microplate luminometer, SpectraMax L (Molecular Devices, Sunnyvale, Calif.).
Luminescence induction of fold changes was calculated by the ratio of relative light unit (RLU) (induced) to RLU (no antibody control). EC50 was determined by plotting x (concentration in μg/mL)−y (the induction of fold change) and fitting the data in a 4PL nonlinear regression model by PRISM 6 Software.
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5
FcγRIIIa-Mediated ADCC Bioassay
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