Production and characterization of monoclonal antibodies to synthetic human ferroportin peptide

Background. Ferroptosis is  a  type of  non-apoptotic iron-dependent form of  regulated cell death. Ferroportin is the only exporter of iron from cells, however, the involvement of ferroportin in cell ferroptosisis poorly understood. Activation or blocking of  ferroptosis pathways may provide a  therapeutic strategy for the  treatment of a  number of diseases. Control of ferroportin expression will make it possible to select a more effective tumor therapy. Obtaining monoclonal antibodies to human ferroportin will help to further study its role in ferroptosis of tumor cells.

Aim. Obtaining and characterization of monoclonal antibodies to human ferroportin.

Materials and methods. Mouse monoclonal antibodies against a synthetic peptide corresponding to the ferroportin protein region were obtained using hybridomic technology. The peptide was synthesized by the method of solid-phase automatic synthesis.

Results. A 15-membered peptide with the sequence ELKQLNLHKDTEPKP (ELK) was selected and synthesized. Conjugates of the synthetic peptide with carrier proteins were obtained. 4 stable hybridomic clones were obtained, and samples of monoclonal antibodies were obtained. The samples were successfully tested using methods of flow cytometry, enzyme immunoassay, and polyacrylamide gel electrophoresis.

Conclusion. Four antibodies against ferroportin binding the ELK peptide were obtained and characterized. The antibodies can be used in further studies of iron transport in tumor cells.

1. Holohan C., Van Schaeybroeck S., Longley D., Johnston P.G. Cancer drug resistance: an evolving paradigm. Nat Rev Cancer 2013;13(10):714–26. DOI: 10.1038/nrc3599

2. Okada H., Mak T.W. Pathways of apoptotic and non-apoptotic death in tumour cells. Nat Rev Cancer 2004;4(8):592–603. DOI: 10.1038/nrc1412

3. Dixon S.J., Lemberg K.M., Lamprecht M.R. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 2012;149(5):1060–72. DOI: 10.1016/j.cell.2012.03.042

4. Galluzzi L., Vitale I., Aaronson S.A. et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ 2018;25(3):486–541. DOI: 10.1038/s41418-017-0012-4

5. Halliwell B. The wanderings of a free radical. Free Radic Biol Med 2009;46(5):531–42. DOI: 10.1016/j.freeradbiomed.2008.11.008

6. Lisova A.E., Baranovskiy A.G., Morstadt L.M. et al. The iron-sulfur cluster is essential for DNA binding by human DNA polymerase ε. Sci Rep 2022;12(1):17436. DOI: 10.1038/s41598-022-21550-4

7. Hassannia B., Wiernicki B., Ingold I. et al. Nano-targeted induction of dual ferroptotic mechanisms eradicates high-risk neuroblastoma. J Clin Invest 2018;128(8):3341–55. DOI: 10.1172/JCI99032

8. Li J., Cao F., Yin H.L. et al. Ferroptosis: past, present and future. Cell Death Dis 2020;11(2):88. DOI: 10.1038/s41419-020-2298-2

9. Hassannia B., Vandenabeele P., Vanden Berghe T. Targeting ferroptosis to iron out cancer. Cancer Cell 2019;35(6):830–49. DOI: 10.1016/j.ccell.2019.04.002

10. Vartanian A.A. Iron metabolism, ferroptosis and cancer. Rossijskij bioterapevticeskij zurnal = Russian Journal of Biotherapy 2017;16(3):14–20. (In Russ.). DOI: 10.17650/1726-9784-2017-16-3-14-20

11. Uniprot. URL: https://www.uniprot.org/uniprotkb/Q9NP59/entry.

12. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256(5517):495–7. DOI: 10.1038/256495a0

13. Engvall E., Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971;8(9):871–4. DOI: 10.1016/0019-2791(71)90454-x

14. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680–5. DOI: 10.1038/227680a0