Figure 4.

Application of TrueRepertoire™ to a phage-displayed scFv library targeting hHGF protein. (a) Comparison of the frequencies of a heavy-chain sequence of the identified clones between the NGS and TrueRepertoire™ results. To confirm that the clonal frequencies of the clones identified through TrueRepertoire™ reflect the real distribution of the library, the frequencies of the heavy-chain sequence were calculated and compared with those from NGS. (b) Groups of the identified clones. To clarify the distribution of the library, the clones were categorized into three groups, major, minor, and rare, according to the clonal frequency of the clones. The major group was denoted in red, the minor group in green, and the rare group in blue. Positive clones having binding activity to hHGF of each group were denoted in darker colors. (c) Composition of the clonal frequencies of the groups and the number of unique clones in each group. The clonal frequency of the groups refers to the sum of clonal frequencies of the clones in each group. (d) Binding activity test of the clones by ELISA. A total of 384 clones were retrieved and then tested for their binding activity to hHGF protein. All clones in the major and minor groups were retrieved and then tested for their binding activity. In the rare group, 254 clones were selected randomly from the total number of clones in the group and then tested for their binding activity. Among the tested 384 clones, 374 clones were confirmed to be positive clones with binding activity. (e) The expected composition of total positive clones in the library. The expected number of positive clones in the rare group was estimated based on the positive rate from the previous ELISA test. (f) The results of the sampling simulation. To determine how the number of positive clones and the composition of the positive clones changes as the throughput of TrueRepertoire™ increases, a sampling simulation was conducted.