Description
Assay Performance Data
Differential binding of anti-hCD20 isotypes to the high-affinity FcγRI measured with the Lumit® FcγRI Binding Immunoassay. A representative binding curve for anti-hCD20 IgG1, IgG2, IgG3 and IgG4 interacting with FcγRI is shown as normalized luminescence. The maximal bioluminescent signal generated in the absence of analyte (0nM antibody) was defined as 100%, and the signal at each antibody concentration was expressed as a percentage of this maximum. Data were fit with a four-parameter logistic regression model using 1/y² weighting to determine IC₅₀ values.
FcγRIIIa (V158) binding of rituximab biosimilars measured with the Lumit® FcγRIIIa (V158) Binding Immunoassay. A representative binding curve for Control Anti-CD20 IgG1, ublituximab and obinutuzumab is plotted as normalized luminescence. The maximal bioluminescent signal observed in the absence of analyte (0nM antibody) was defined as 100%, and the signal at each antibody concentration was expressed as a percentage of this maximum. Data were fitted to a four-parameter logistic regression model with 1/y² weighting to calculate apparent IC₅₀ values.
Effect of H₂O₂-mediated oxidation on FcγRIIa (H131) binding of human IgG3 measured with the Lumit® FcγRIIa Binding Immunoassay. Human IgG3 was added to H₂O₂ solutions to yield final peroxide concentrations of 3%, 1%, 0.3% or 0% (control). Samples were exposed for 2 hours at RT followed by removal of residual H₂O₂. Serial dilutions of each treated antibody were assayed, and raw luminescence (RLU) was plotted against IgG3 concentration. Curves were fitted with a four-parameter logistic regression model employing 1/y² weighting to determine apparent IC₅₀ values.
Effect of Fc glycan removal on FcγRI binding measured with the Lumit® FcγRI Binding Immunoassay. A representative binding curve is shown for native human IgG and PNGase F–deglycosylated IgG. Deglycosylation was performed overnight at 37°C (4mg/ml⁻¹ IgG; 1ml PNGase F per 20mg of IgG). The maximal bioluminescent signal obtained in the absence of analyte (0nM IgG) was defined as 100%, and the signal at each IgG concentration was expressed as a percentage of this maximum. Data were fitted to a four-parameter logistic regression equation with 1/y² weighting to calculate apparent IC₅₀ values.
Lumit® FcγRIIb Binding Immunoassay. A representative standard curve of FcγR Control Antibody is plotted as normalized data. Normalized luminescence is calculated by setting the maximum bioluminescent signal (observed in the absence of an analyte) as 100% and expressing the signal in the presence of an analyte as a percentage of this maximum. Data were fitted to a four-parameter logistic regression equation with 1/y 2 weighting.
