Single-Domain Nanobodies for Determination of Conformational Changes in Transferrin and Their Use in Fluorescent Polarization Immunoassay

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

A method for the synthesis of aTf1 and aTf2 nanobodies conjugates, previously obtained for human holo- and apo-transferrin (Tf) with fluorescein isothiocyanate (FITC), is proposed. The conjugates were used as tracers for the fluorescence polarization immunoassay (FPIA) method with nanobodies. Optimal concentrations of FITC-aTf1 and FITC-aTf2 conjugates (2.5–5 nM) were selected. Binding kinetics of FITC-aTf1 and FITC-aTf2 with holo- and apo-Tf was studied. A complete binding of FITC-aTf1 and FITC-aTf2 conjugates with holo- and apo-Tf was observed after 15 and 5 min of incubation, respectively. The equilibrium dissociation constants of FITC-aTf1*holo-Tf and FITC-aTf2*apo-Tf complexes were determined, which amounted to 30.7 ± 0.3 and 15.3 ± 0.2 nM. A high specificity of analysis was verified by the incubation of FITC-aTf1 and FITC-aTf2 conjugates with other human proteins, lactoferrin, serum albumin, lysozyme. A high affinity of the conjugates FITC-aTf1 and FITC-aTf2 to holo- and apo-Tf was also shown. The synthesized FITC-aTf1 and FITC-aTf2 conjugates have potential for determining transferrin various conformations in human physiological fluids. Thus, this work demonstrates the possibility of determining two forms of transferrin in human physiological fluids using the FPIA method, which may have diagnostic value, and the use of a portable fluorescence analyzer will allow this analysis to be carried out outside the walls of specialized laboratories.

Full Text

Restricted Access

About the authors

L. I. Mukhametova

Lomonosov Moscow State University

Author for correspondence.
Email: liliya106@mail.ru

Department of Chemistry

Russian Federation, Leninskie gory 1/3, Moscow, 119234

S. A. Eremin

Lomonosov Moscow State University

Email: liliya106@mail.ru

Department of Chemistry

Russian Federation, Leninskie gory 1/3, Moscow, 119234

I. V. Mikhura

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: liliya106@mail.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

O. S. Goryainova

Institute of Gene Biology Russian Academy of Sciences

Email: liliya106@mail.ru
Russian Federation, ul. Vavilova 34/5, Moscow, 119334

A. M. Sachko

Institute of Gene Biology Russian Academy of Sciences

Email: liliya106@mail.ru
Russian Federation, ul. Vavilova 34/5, Moscow, 119334

T. I. Ivanova

Institute of Gene Biology Russian Academy of Sciences

Email: liliya106@mail.ru
Russian Federation, ul. Vavilova 34/5, Moscow, 119334

S. V. Tillib

Institute of Gene Biology Russian Academy of Sciences

Email: tillib@genebiology.ru
Russian Federation, ul. Vavilova 34/5, Moscow, 119334

References

  1. Tatsumi Y., Yano M., Wakusawa S., Miyajima H., Ishikawa T., Imashuku S., Takano A., Nihei W., Kato A., Kato K., Hayashi H., Yoshioka K., Hayashi K. // J. Clin. Transl. Hepatol. 2024. V. 12. P. 346–356. https://doi.org/10.14218/JCTH.2023.00290
  2. Sarkar J., Potdar A.A., Saidel G.M. // PLoS Comput. Biol. 2018. V. 14. e1006060. https://doi.org/10.1371/journal.pcbi.1006060
  3. Schreiner O.D., Schreiner T.G. // Front. Aging. 2023. V. 4. P. 1234958. https://doi.org/10.3389/fragi.2023.1234958
  4. Tandara L., Salamunic I. // Biochem. Med. (Zagreb). 2012. V. 22. P. 311–328. https://doi.org/10.11613/bm.2012.034
  5. Wally J., Halbrooks P.J., Vonrhein C., Rould M.A., Everse S.J., Mason A.B., Buchanan S.K. // J. Biol. Chem. 2006. V. 281. P. 24934–24944. https://doi.org/10.1074/jbc.M604592200
  6. Baker E.N., Lindley P.F. // J. Inorg. Biochem. 1992. V. 47. P. 147–160. https://doi.org/10.1016/0162-0134(92)84061-q
  7. Ponzini E., Scotti L., Grandori R., Tavazzi S., Zambon A. // Invest. Ophthalmol. Vis. Sci. 2020. V. 61. P. 9. https://doi.org/10.1167/iovs.61.12.9
  8. Withold W., Neumayer C., Beyrau R., Heins M., Schauseil S., Rick W. // Eur. J. Clin. Chem. Clin. Biochem. 1994. V. 32. P. 19–25. https://doi.org/10.1515/cclm.1994.32.1.19
  9. Elsayed M.E., Sharif M.U., Stack A.G. // Adv. Clin. Chem. 2016. V. 75. P. 71–97. https://doi.org/10.1016/bs.acc.2016.03.002
  10. Muñoz M., García-Erce J.A., Remacha Á.F. // J. Clin. Pathol. 2011. V. 64. P. 287–296. https://doi.org/10.1136/jcp.2010.086991
  11. Szőke D., Panteghini M. // Clin. Chim. Acta. 2012. V. 413. P. 1184–1189. https://doi.org/10.1016/j.cca.2012.04.021
  12. Ivanova T.I., Klabukov I.D., Krikunova L.I., Poluektova M.V., Sychenkova N.I., Khorokhorina V.A., Vorobyev N.V., Gaas M.Y., Baranovskii D.S., Goryainova O.S., Sachko A.M., Shegay P.V., Kaprin A.D., Tillib S.V. // J. Clin. Med. 2022. V. 11. P. 7377. https://doi.org/10.3390/jcm11247377
  13. Baringer S.L., Neely E.B., Palsa K., Simpson I.A., Connor J.R. // Fluids Barriers CNS. 2022. V. 19. P. 49. https://doi.org/10.1186/s12987-022-00345-9
  14. Yang N., Zhang H., Wang M., Hao Q., Sun H. // Sci. Rep. 2012. V. 2. P. 999. https://doi.org/10.1038/srep00999
  15. Baringer S.L., Palsa K., Spiegelman V.S., Simpson I.A., Connor J.R. // J. Biomed. Sci. 2023. V. 30. P. 36. https://doi.org/10.1186/s12929-023-00934-2
  16. Bassett M.L., Halliday J.W., Ferris R.A., Powell L.W. // Gastroenterology. 1984. V. 87. P. 628–633.
  17. MacPhail A.P., Mandishona E.M., Bloom P.D., Paterson A.C., Rouault T.A., Gordeuk V.R. // S. Afr. Med. J. 1999. V. 89. P. 966–972.
  18. Yamanishi H., Iyama S., Yamaguchi Y., Kanakura Y., Iwatani Y. // Clin. Chem. 2003. V. 49. P. 175–178. https://doi.org/10.1373/49.1.175
  19. Huebers H.A., Eng M.J., Josephson B.M., Ekpoom N., Rettmer R.L., Labbé R.F., Pootrakul P., Finch C.A. // Clin. Chem. 1987. V. 33. P. 273–277.
  20. Lopez A., Cacoub P., Macdougall I.C., PeyrinBiroulet L. // Lancet. 2016. V. 387. P. 907–916. https://doi.org/10.1016/S0140-6736(15)60865-0
  21. Camaschella C. // Blood Rev. 2017. V. 31. P. 225–233. https://doi.org/10.1016/j.blre.2017.02.004
  22. Yamanishi H., Kimura S., Iyama S., Yamaguchi Y., Yanagihara T. // Clin. Chem. 1997. V. 43. P. 2413– 2417. https://doi.org/10.1093/clinchem/43.12.2413
  23. Gambino R., Desvarieux E., Orth M., Matan H., Ackattupathil T., Lijoi E., Wimmer C., Bower J., Gunter E. // Clin. Chem. 1997. V. 43. P. 2408–2412. https://doi.org/10.1093/clinchem/43.12.2408
  24. Strzelak K., Rybkowska N., Wiśniewska A., Koncki R. // Anal. Chim. Acta. 2017. V. 995. P. 43–51. https://doi.org/10.1016/j.aca.2017.10.015
  25. Eleftheriadis T., Liakopoulos V., Antoniadi G., Stefanidis I. // Ren. Fail. 2010. V. 32. P. 1022–1023. https://doi.org/10.3109/0886022X.2010.502609
  26. Kitsati N., Liakos D., Ermeidi E., Mantzaris M.D., Vasakos S., Kyratzopoulou E., Eliadis P., Andrikos E., Kokkolou E., Sferopoulos G., Mamalaki A., Siamopoulos K., Galaris D. // Haematologica. 2015. V. 100. P. e80–e83. https://doi.org/10.3324/haematol.2014.116806
  27. Angoro B., Motshakeri M., Hemmaway C., Svirskis D., Sharma M. // Clin. Chim. Acta. 2022. V. 531. P. 157–167. https://doi.org/10.1016/j.cca.2022.04.004
  28. Agarwal R. // Kidney Int. 2004. V. 66. P. 1139–1144. https://doi.org/10.1111/j.1523-1755.2004.00864.x
  29. DeGregorio-Rocasolano N., Martí-Sistac O., Ponce J., Castelló-Ruiz M., Millán M., Guirao V., García-Yébenes I., Salom J.B., Ramos-Cabrer P., Alborch E., Lizasoain I., Castillo J., Dávalos A., Gasull T. // Redox Biol. 2018. V. 15. P. 143–158. https://doi.org/10.1016/j.redox.2017.11.026
  30. Drain P.K., Hyle E.P., Noubary F., Freedberg K.A., Wilson D., Bishai W.R., Rodriguez W., Bassett I.V. // Lancet Infect. Dis. 2014. V. 14. P. 239–249. https://doi.org/10.1016/S1473-3099(13)70250-0
  31. Karim K., Lamaoui A., Amine A. // J. Pharm. Biomed. Anal. 2023. V. 225. P. 115207. https://doi.org/10.1016/j.jpba.2022.115207
  32. Arbabi Ghahroudi M., Desmyter A., Wyns L., Hamers R., Muyldermans S. // FEBS Lett. 1997. V. 414. P. 521–526. https://doi.org/10.1016/S0014-5793(97)01062-4
  33. Muyldermans S. // Annu. Rev. Biochem. 2013. V. 82. P. 775–797. https://doi.org/10.1146/annurev-biochem-063011092449
  34. Sockolosky J.T., Dougan M., Ingram J.R., Ho C.C., Kauke M.J., Almo S.C., Ploegh H.L., Garcia K.C. // Proc. Nat. Acad. Sci. USA. 2016. V. 113. P. E2646– E2654. https://doi.org/10.1073/pnas.1604268113
  35. Mukhametova L.I., Eremin S.A., Arutyunyan D.A., Goryainova O.S., Ivanova T.I., Tillib S.V. // Biochemistry (Moscow). 2022. V. 87. P. 1679–1688. https://doi.org/10.1134/s0006297922120227
  36. Dumoulin M., Conrath K., Van Meirhaeghe A., Meersman F., Heremans K., Frenken L.G., Muyldermans S., Wyns L., Matagne A. // Protein Sci. Publ. Protein Soc. 2002. V. 11. P. 500–515. https://doi.org/10.1110/ps.34602
  37. Xu L., Song X., Jia L. // Biotechnol. Appl. Biochem. 2017. V. 64. P. 895–901. https://doi.org/10.1002/bab.1544
  38. Jovčevska I., Muyldermans S. // BioDrugs Clin. Immunother. Biopharm. Gene Ther. 2020. V. 34. P. 11–26. https://doi.org/10.1007/s40259-019-00392-z
  39. Khodabakhsh F., Behdani M., Rami A., KazemiLomedasht F. // Int. Rev. Immunol. 2018. V. 37. P. 316– 322. https://doi.org/10.1080/08830185.2018.1526932
  40. Mei Y., Chen Y., Sivaccumar J.P., An Z., Xia N., Luo W. // Front. Pharmacol. 2022. V. 13. P. 963978. https://doi.org/10.3389/fphar.2022.963978
  41. Bao G., Tang M., Zhao J., Zhu X. // EJNMMI Res. 2021. V. 11. P. 6. https://doi.org/10.1186/s13550-021-00750-5
  42. Тиллиб С.В., Горяйнова О.С., Сачко А.М., Иванова Т.И. // Acta Naturae. 2022. Т. 14. C. 98–102. https://doi.org/10.32607/actanaturae.11663
  43. Сачко А.М., Горяйнова О.С., Иванова Т.И., Николаева И.Ю., Тарнопольская М.Е., Бычков А.Ю., Гаас М.Я., Воробьев Н.В., Каприн А.Д., Шегай П.В., Тиллиб С.В. // Биохимия. 2023. Т. 88. С. 1352–1365. https://doi.org/10.31857/S0320972523080055
  44. Yu L., Zhong M., Wei Y. // Anal. Chem. 2010. V. 82. P. 7044–7048. https://doi.org/10.1021/ac100543e

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. Binding kinetics of the conjugates FITC-tf2 (1) and FIT CaT f1 (2) 2 nM capo- and halo-Tf, respectively. The final concentrations of apo- and holo-Tf are 3 micrograms/ml; pH 7.4, 25°C.

Download (100KB)
Indexing metadata
3. Fig. 2. Variation of the FP signal depending on the concentration of ap-Tf (1) and halo-Tf(2) in the presence of 2.5 nM FITC-tf2 (1) and FIT CaT f1 (2); pH 7.4, 25°C.

Download (98KB)
Indexing metadata
4. 3. The ratio of the concentration of antibody-bound tracer Cx to the initial concentration of tracer FITC-aTf0-(Fb) on the concentration of apo-Tf (1) and holo-Tf (2) at constant concentrations of fluorescently labeled nanobodies FITC-aTf2 (1) and FITC-aTf1 (2) 2 nM; pH 7.4, 25°C.

Download (100KB)
Indexing metadata
5. Fig. 4. Specificity of FIT CaT f1 and FITC-aTf2 nanobodies in interaction with other proteins; pH 7.4, 25°C.

Download (102KB)
Indexing metadata
6. Fig. 5. Linear range of calibration dependences for the determination of apo-Tf (1) and holo-Tf (2); pH 7.4, 25°C.

Download (111KB)
Indexing metadata

Copyright (c) 2025 Russian Academy of Sciences