Figure 2

Comparative binding assays of soluble SU proteins of FeLV-A/61E or FeLV-945. A. A representative histogram is shown from a comparative flow cytometric binding assay demonstrating the binding activity of FeLV-A/61E SU (61E; gray shaded) or FeLV-945 SU (945; black shaded). Soluble SU proteins were quantified precisely using anti-SU antibody C11D8. Feline 3201 cells were incubated with equivalent mass amounts of either SU protein for one hour, followed by incubation with C11D8 antibody to detect the surface-bound viral SU proteins and then with an Alexa Fluor 488-conjugated secondary antibody. Negative controls (open histograms) included cell supernatants of transfections with the empty expression vector, pCS2/Ctrl, and each SU with isotype control antibody. B. Chemiluminescent western blot analysis of equivalent mass amounts of FeLV-A/61E and FeLV-945 SU proteins using C11D8 antibody as probe is shown to validate the precision of the infrared quantification. Negative control was supernatants of cells transfected with pCS2/Ctrl. C - D. Geometric mean fluorescence of replicate binding assays performed using four independently generated and quantified batches of FeLV-A/61E and FeLV-945 SU protein on either feline 3201 cells (C) or murine MDTF/H2 cells (D) which express the FeLV-A receptor. Supernatant of mock- or pCS2/Ctrl-transfected cells were used as a negative control. The mean of replicate experiments is represented (horizontal bar). Asterisk indicates statistical significance (*; p < 0.001). E. Flow cytometric binding assays performed exactly as in (A) except that analysis was performed using an antibody to detect the HA tag at the C-terminus of soluble SU proteins. Shown are a representative histogram (left), anti-HA chemiluminescent western blot analysis of equivalent mass amounts of SU proteins to validate quantification (inset), and geometric mean fluorescence of replicate binding assays (right; p < 0.001). Negative controls included either SU protein with isotype control antibody (open histograms).