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. 2022 May 6;23(9):5208. doi: 10.3390/ijms23095208

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© 2022 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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BER1x3-derived clones from libraries BER1x3_4NNK and BER1x3_5NNK, selected with mammalian cells-derived CD59 antigen: (a) ELISA response of rituximab (RX)-based mAb² molecules to HEK-expressed CD59 and mouse serum albumin as a control antigen; (b) Complement-dependent cytotoxicity (CDC) assay of RX-based bispecific antibodies on Raji cells, compared with parental clone RX-BER1x3, in 2-fold dilutions starting from 5 nM, decreasing concentrations are presented from dark to light blue (sample size n = 2, mean and S.D. are shown); (c) Profiles of RX-derivates in SEC-HPLC in native conditions (MWS: molecular weight standard); (d) Biolayer interferometry-measured response of RX-BER5-1-3 to bacterially expressed and refolded CD59 (left panel) and mammalian cells expressed CD59 (right panel), in 2-fold dilutions starting from 300 nM; (e) Comparison of the CDC effect of RX and RX-BER5-1-3 on Raji cells (left panel), ARH-77 cells (central panel) and Daudi cells (right panel) (sample size n = 3 for experiments done with Raji and n = 2 for ARH-77 and Daudi, one-way ANOVA was used to determine the significance (n.s. (not significant) 0.05 ≤ p, * 0.01 ≤ p < 0.05, ** 0.001 ≤ p < 0.01, *** 0.0001 ≤ p < 0.001), mean and S.D. are shown; (f) SK-BR-3 cell surface binding of RX-based mAb² molecules at 2 µM concentration (left panel) and the CDC (in)activity of RX and the mAb² derivates on this cell line, sample size n = 2 (right panel). Mean and S.D. are presented.

BER1x3-derived clones from libraries BER1x3_4NNK and BER1x3_5NNK, selected with mammalian cells-derived CD59 antigen: (a) ELISA response of rituximab (RX)-based mAb² molecules to HEK-expressed CD59 and mouse serum albumin as a control antigen; (b) Complement-dependent cytotoxicity (CDC) assay of RX-based bispecific antibodies on Raji cells, compared with parental clone RX-BER1x3, in 2-fold dilutions starting from 5 nM, decreasing concentrations are presented from dark to light blue (sample size n = 2, mean and S.D. are shown); (c) Profiles of RX-derivates in SEC-HPLC in native conditions (MWS: molecular weight standard); (d) Biolayer interferometry-measured response of RX-BER5-1-3 to bacterially expressed and refolded CD59 (left panel) and mammalian cells expressed CD59 (right panel), in 2-fold dilutions starting from 300 nM; (e) Comparison of the CDC effect of RX and RX-BER5-1-3 on Raji cells (left panel), ARH-77 cells (central panel) and Daudi cells (right panel) (sample size n = 3 for experiments done with Raji and n = 2 for ARH-77 and Daudi, one-way ANOVA was used to determine the significance (n.s. (not significant) 0.05 ≤ p, * 0.01 ≤ p < 0.05, ** 0.001 ≤ p < 0.01, *** 0.0001 ≤ p < 0.001), mean and S.D. are shown; (f) SK-BR-3 cell surface binding of RX-based mAb² molecules at 2 µM concentration (left panel) and the CDC (in)activity of RX and the mAb² derivates on this cell line, sample size n = 2 (right panel). Mean and S.D. are presented.