Mapping the Binding Sites of CA125-Specific Antibodies on a Revised Molecular Model of MUC16
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. CA125 Recombinant Protein Expression and Purification
2.2. Western Blot
2.3. Mass Spectrometry
2.3.1. S-Trap Digestion
2.3.2. In-Gel Digestion
2.3.3. Mass Spectrometry Data Acquisition
2.3.4. Mass Spectrometry Analysis
2.4. ELISA
2.5. SPR Materials
2.6. M11 Stock Concentration Determination
2.7. SPR Binding Kinetics Assay Sample Preparation
2.8. SPR Instrumental Protocol
2.9. SPR Binding Kinetics Assessment
3. Results
3.1. Recombinant Tandem Repeat Protein Expression and Verification
3.2. ELISA
3.3. SPR
3.4. Comparison of Analytical Methods (ELISA and SPR)
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Repeat | Sequence | Coverage |
---|---|---|
R1 | ATVPFMVPFTLNFTITNLQYEEDMRHPGSRKFNATERELQGLLKPLFRNSSLEYLYSGCRLAS LRPEKDSSATAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFT HRSSMPTTSTPGTSTVDVGTSGTPSSSPSPT | 100% |
R2 | TAGPLLMPFTLNFTITNLQYEEDMRRTGSRKFNTMESVLQGLLKPLFKNTSVGPLYSGCRLT LLRPEKDGAATGVDAICTHRLDPKSPGLNREQLYWELSKLTNDIEELGPYTLDRNSLYVNGF THQSSVSTTSTPGTSTVDLRTSGTPSSLSSPTIM | 100% |
R3 | AAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVGPLYSGCRLTS LRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYTLDRNSLYVNGFT HRTSVPTTSTPGTSTVDLGTSGTPFSLPSPA | 97% |
R4 | TAGPLLVLFTLNFTITNLKYEEDMHRPGSRKFNTTERVLQTLLGPMFKNTSVGLLYSGCRLT LLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFT HWIPVPTSSTPGTSTVDLGSGTPSSLPSPT | 99% |
R5 | TAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLLGPMFKNTSVGPLYSGCRLT LLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNG FTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPT | 100% |
R6 | SAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLT LLRPEKNGAATGMDAICSHRLDPKSPGLNREQLYWELSQLTHGIKELGPYTLDRNSLYVNG FTHRSSVAPTSTPGTSTVDLGTSGTPSSLPSPT | 95% |
R7 | TAVPLLVPFTLNFTITNLQYGEDMRHPGSRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLIS LRSEKDGAATGVDAICTHHLNPQSPGLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNG FTHRSSGLTTSTPWTSTVDLGTSGTPSPVPSPT | 100% |
R8 | TAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLQGLLSPIFKNSSVGPLYSGCRLTSL RPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPYSLDRDSLYVNGF THQNSVPTTSTPGTSTVYWATTGTPSSFPGHT | 100% |
R9 | EPGPLLIPFTFNFTITNLHYEENMQHPGSRKFNTTERVLQGLLTPLFKNTSVGPLYSGCRLTLL RPEKHEAATGVDTICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNP WSSVPTTSTPGTSTVHLATSGTPSSLPGHT | 100% |
R10 | APVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLL RPEKHGAATGVDAICTLRLDPTGPGLDRERLYWELSQLTNSVTELGPYTLDRDSLYVNGFT HRSSVPTTSIPGTSAVHLETSGTPASLPGHT | 100% |
R11 | APGPLLVPFTLNFTITNLQYEEDMRHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTL LRPEKRGAATGVDTICTHRLDPLNPGLDREQLYWELSKLTRGIIELGPYLLDRGSLYVNGFT HRNFVPITSTPGTSTVHLGTSETPSSLPRPI | 100% |
R12 | VPGPLLVPFTLNFTITNLQYEEAMRHPGSRKFNTTERVLQGLLRPLFKNTSIGPLYSSCRLTLL RPEKDKAATRVDAICTHHPDPQSPGLNREQLYWELSQLTHGITELGPYTLDRDSLYVDGFT HWSPIPTTSTPGTSIVNLGTSGIPPSLPETT | 100% |
R13 | ATGPLLVPFTLNFTITNLQYEENMGHPGSRKFNITESVLQGLLKPLFKSTSVGPLYSGCRLTLL RPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQ RSSVPTTSTPGTFTVQPETSETPSSLPGPT | 88% |
R14 | ATGPVLLPFTLNFTIINLQYEEDMHRPGSRKFNTTERVLQGLLMPLFKNTSVSSLYSGCRLTL LRPEKDGAATRVDAVCTHRPDPKSPGLDRERLYWKLSQLTHGITELGPYTLDRHSLYVNGF THQSSMTTTRTPDTSTMHLATSRTPASLSGPT | N/A |
R15 | TASPLLVLFTINFTITNLRYEENMHHPGSRKFNTTERVLQGLLRPVFKNTSVGPLYSGCRLTL LRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTLDRDSLYVNGF TQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPS | 99% |
R16 | AASPLLVLFTLNFTITNLRYEENMQHPGSRKFNTTERVLQGLLRSLFKSTSVGPLYSGCRLTL LRPEKDGTATGVDAICTHHPDPKSPRLDREQLYWELSQLTHNITELGPYALDNDSLFVNGF THRSSVSTTSTPGTPTVYLGASKTPASIFGPS | 76% |
R17 | AASHLLILFTLNFTITNLRYEENMWPGSRKFNTTERVLQGLLRPLFKNTSVGPLYSGCRLTLL RPEKDGEATGVDAICTHRPDPTGPGLDREQLYLELSQLTHSITELGPYTLDRDSLYVNGF THRSSVPTTSTGVVSEE | 100% |
R18 | PFTLNFTINNLRYMADMGQPGSLKFNITDNVMQHLLSPLFQRSSLGARYTGCRVIALRSVK NGAETRVDLLCTYLQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGPD EPPTTPKPATTFLPPLSEATT | N/A |
R19 | AMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRPLFQKSSMGPFYLGCQLIS LRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHGVTQLGFYVLDRDSLFINGY APQNLSIRGEYQINFHIVNWNLSNPDPT | N/A |
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Wang, C.-W.; Srivastava, A.; Hanson, E.K.; McEntee, C.R.; Nair, T.; March, J.C.; Whelan, R.J. Mapping the Binding Sites of CA125-Specific Antibodies on a Revised Molecular Model of MUC16. Cancers 2025, 17, 1458. https://doi.org/10.3390/cancers17091458
Wang C-W, Srivastava A, Hanson EK, McEntee CR, Nair T, March JC, Whelan RJ. Mapping the Binding Sites of CA125-Specific Antibodies on a Revised Molecular Model of MUC16. Cancers. 2025; 17(9):1458. https://doi.org/10.3390/cancers17091458
Chicago/Turabian StyleWang, Chien-Wei, Anubhuti Srivastava, Eliza K. Hanson, Caitlin R. McEntee, Trisha Nair, Jane C. March, and Rebecca J. Whelan. 2025. "Mapping the Binding Sites of CA125-Specific Antibodies on a Revised Molecular Model of MUC16" Cancers 17, no. 9: 1458. https://doi.org/10.3390/cancers17091458
APA StyleWang, C.-W., Srivastava, A., Hanson, E. K., McEntee, C. R., Nair, T., March, J. C., & Whelan, R. J. (2025). Mapping the Binding Sites of CA125-Specific Antibodies on a Revised Molecular Model of MUC16. Cancers, 17(9), 1458. https://doi.org/10.3390/cancers17091458