MOESM1 of Development of a novel human phage display-derived anti-LAG3 scFv antibody targeting CD8+ T lymphocyte exhaustion

Additional file 1: Figure S3DII. Characterization of the anti-LAG3 scFvF7 (II). A. ELISA conducted in parallel on intact recombinant LAG3 protein and heat-denatured LAG3 protein (by boiling for 5 min). The plate was coated with 0.5 μg of antigen per microwell (GO, intact recombinant LAG3 and heat-stressed recombinant LAG3). After blocking step (2% MPBS for two hours at r.t.) wells were incubated for 2 h at r.t. with 50 μL of scFvF7 (25 μg/mL) together with anti-flag M2 Ab (2.5 μg/mL, Sigma) and HRP- conjugated anti-mouse Ab (5 μg/mL, Dako). A mouse anti-6 his mAb (which recognizes the 6-histidines tag at C terminal end of LAG3 protein)(Serotec) and commercial mouse anti-LAG3 mAb 17B4 (that recognizes the 30 aa extra-loop of the first N-terminal D1 domain of human LAG3)(EnzoLab) were used as positive controls. All Abs were resuspended in 2% MPBS. O.D.: optical density. B. Western blotting assay with LAG3 under reducing and non-reducing conditions. 0.5 μg of glucose oxidase (GO) and of recombinant LAG3 proteins (the latter under three different conditions, namely: -DTT (reducing agent)/−boiling, −DTT/+boiling 5 min, +DTT/+boiling 5 min) were loaded as specified in replicate wells on a 12% SDS-PAGE and transferred to filter paper. Portions from the filter were then incubated with the indicated primary antibodies. An 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). Arrows indicate relevant signals. The different molecular weights observed for LAG3 are obviously attributable to the impact of the different experimental conditions (non-reducing and reducing) on the SDS-PAGE separation. Molecular markers in kilodaltons (kDa) are reported on the right. The reactivity of the supernatants (scFvGO and scFvF7) used was previously checked in ELISA (bottom). (PPTX 125 kb)