Fig. 4: CDC enhancement is also observed with different hIgG isotypes and another CD20 epitope.

C1q binding was measured by ELISA. ELISA plates were coated with hIgG µtp constructs at various concentrations and purified human C1q (2 μg/ml) added. Bound C1q was detected with a goat-anti-C1q, followed by an anti-goat-HRP-conjugated antibody. Data show absorbance at 450 nm. C1q cell recruitment was assessed by opsonising Ramos cells with hIgG µtp constructs, followed by incubation with 2 μg/ml human C1q. Deposition of C1q was analysed with an anti-C1q-FITC antibody. CDC-induced cell death was assessed by opsonising Ramos cell with 10–0.15 µg/ml hIgG µtp constructs and incubated with NHS (20 % V/V). Cell death was examined as the percentage of PI-positive cells by flow cytometry. Results are shown for a rituximab hIgG2, b rituximab hIgG4, and c BHH2 hIgG1 µtp constructs. d Ramos cells were opsonised with daratumumab (anti-CD38) hIgG1 μtp mAb at 10 μg/ml and binding measured by secondary anti-human Fc-APC-labelled antibody. Solid grey histograms indicate trastuzumab hIgG isotype control mAb. e Antibody binding (MFI) over a concentration range (n = 1). f CDC-induced cell death was assessed for daratumumab μtp antibodies after opsonisation of Ramos cells. Individual data points and mean shown from biologically independent experiments (n = 3).