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. 2023 Aug 11;7:74. doi: 10.1038/s41698-023-00423-7

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Graphical representation of mAb5E6 epitope on surface tethered carboxy-terminal (Cter) domain of MUC16. b Schematic representation of ch5E6 generation by grafting murine V_H and V_L regions on human IgG1 FcH and FcL harboring plasmids and co-transfecting in ExpiCHO cells. c ELISA for binding of chimeric mb5E6 to recombinant MUC16 C-ter protein (rMUC16-Cter) and its comparison to parent murine version mAb5E6.Y-axis, binding as absorbance measurement at 450 nm; X-axis, mAb concentrations; d Immunoblot analysis of ch5E6 with E.coli expressed, and MIA PaCa-2 cells transfected rMUC16-C ter protein lysates. e Real-time binding analysis of anti-MUC16 murine (m5E6) and chimeric mAb5E6 (ch5E6) to rMUC16-Cter protein using SPR (Surface Plasmon Resonance). Different concentrations of rMUC16-Cter from 0 nm to 1 µM were passed over the immobilized chimeric and murine mAb5E6 on sensor chip CMD200m. Ka and Kd values were obtained by the analysis of sensograms on scrubber 2.0 and were used to calculate KD values. Y-axis, amount of bound antibody as resonance units (RU); X-axis, time in seconds. f Immunoblot analysis of conditioned media (CM) prepared from MUC16 expressing PC SW1990, COLO357, and NSCLC H2122 cell lines for determining the binding of ch5E6 to shed from MUC16. Soluble MUC16-specific mAb M11 was used as the positive control, and MIA PaCa-2 cell line-derived CM was used as a negative control. The lysates (40 μg) prepared from these cells were loaded on 6% and probed with both M11 and ch5E6. g, h Immunohistochemical analysis of ch5E6 with MUC16 on tissue microarray (TMA) from patient tumors at various stages of PC (Stage IA; n = 3, IB; n = 32, IIA; n = 16, IIB; n = 14, III; n = 3) and NSCLC (Stage IA; n = 14, IB; n = 10, IIA; n = 6, IIB; n = 11, III; n = 6) and normal adjacent tissues (PC; n = 8, NSCLC; n = 30). Staining with mAb M11 was used to evaluate MUC16 expression in the tissues. A pathologist at UNMC scored the slides. A histoscore (H-score) was calculated by multiplying intensity and positivity. Representative images of ch5E6 binding to different disease stages are shown in parallel. Error bars in 1C indicate SD. Scale bars, 100 µm; *P < 0.05; **P < 0.01, ****P < 0.001. Fig. 1a was “created with Biorender.com”.