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. 2023 Feb 3;14:1060540. doi: 10.3389/fimmu.2023.1060540

Figure 1.

Figure 1

Generation of anti-Syrian hamster PD-L1 clones and their characterization for subcloning. (A) Confirmation of recombinant Syrian hamster PD-L1 expressed in E.coli by SDS-PAGE where M1 indicates protein ladder, BSA (2.00 µg) positive control and R: reducing conditions. Purified recombinant PD-L1 is indicated by the arrow under reducing conditions (~20-25kDa). (B) Detection of PD-L1 on HaPT1 cell line using antiserum of five mice immunized with recombinant PD-L1 by flow cytometry with count, mean and median (FITC+) shown on the left and right respectively. (C) ELISA results of antiserum from give mice after 3rd immunization against recombinant PD-L1. (D) Detection of endogenous PD-L1 on adherent Syrian hamster PBMCs using anti-PD-L1 subclones from hybridoma supernatants (1:100) by flow cytometry. (E) Pearson’s correlation indicating a non-significant relationship between measured concentrations of hybdridoma supernatant and count used in Figure (D). (F) Affinity ranking and epitope binning of anti-PD-L1 subclones as measured by competitive ELISA using recombinant PD-L1. The final two selected clones are highlighted in orange. (G) Detection of endogenous PD-L1 on three Syrian hamster cancer cell lines HT100, HapT1 and HCPC-1 using In-Cell ELISA. The absorbance values were normalized to cell number as measured by Janus Green Whole-Cell Stain. Data and error bars are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA. *p < 0.05, ****p < 0.0001.