Fig. 3

Characterization of the anti-LAG3 scFvF7. a SDS-PAGE analysis on 1 and 5 μg of scFvF7. Molecular markers in kilodaltons (kDa) are reported on the right. b ELISA for the detection of LAG3 antigen carried out with decreasing doses of purified scFvF7. GO: glucose oxidase . O.D.: optical density. The mean values ±SEM are shown as calculated from results of three independent assays. c FACS analysis carried out with scFvF7 on either unstimulated (grey line) or PHA-stimulated (green line) human CD4⁺ T lymphocytes. Inset: results from the same assay carried out with the murine anti-LAG3 17B4 mAb are reported. Four repeats of the experiment gave consistent results. d Binding analysis of scFvF7. 0.5 μg of both recombinant LAG3 and glucose oxidase (GO) proteins were loaded in each replicate well on a 12% SDS-PAGE under reducing condition and transferred to filter paper. Strips from the filter were then incubated with the indicted primary antibodies. Anti-6 his mAb was used as a positive control for LAG3 recombinant protein (which has a 6-histidines tag at its C terminal end). As additional negative controls, two strips were incubated respectively with the anti-Flag antibody and the HRP-conjugated anti-mouse antibody, in order to monitor eventual non-specific signals due to the secondary antibodies used to detect scFv antibodies. The ELISA shown below (performed with the same secondary Abs) is a check for the reactivity of the scFv-containing supernatants (scFvGO and scFvF7) used in the assay. Arrows indicate relevant signals. Molecular markers in kilodaltons (kDa) are reported on the right. A representative of three independent assays is shown