Single-Domain Nanobodies for Determination of Conformational Changes in Transferrin and Their Use in Fluorescent Polarization Immunoassay
- Авторлар: Mukhametova L.I.1, Eremin S.A.1, Mikhura I.V.2, Goryainova O.S.3, Sachko A.M.3, Ivanova T.I.3, Tillib S.V.3
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Мекемелер:
- Lomonosov Moscow State University
- Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
- Institute of Gene Biology Russian Academy of Sciences
- Шығарылым: Том 51, № 2 (2025)
- Беттер: 280-290
- Бөлім: Articles
- URL: https://journals.eco-vector.com/0132-3423/article/view/682738
- DOI: https://doi.org/10.31857/S0132342325020063
- EDN: https://elibrary.ru/LCGAIP
- ID: 682738
Дәйексөз келтіру
Аннотация
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.
Толық мәтін

Авторлар туралы
L. Mukhametova
Lomonosov Moscow State University
Хат алмасуға жауапты Автор.
Email: liliya106@mail.ru
Department of Chemistry
Ресей, Leninskie gory 1/3, Moscow, 119234S. Eremin
Lomonosov Moscow State University
Email: liliya106@mail.ru
Department of Chemistry
Ресей, Leninskie gory 1/3, Moscow, 119234I. Mikhura
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
Email: liliya106@mail.ru
Ресей, ul. Miklukho-Maklaya 16/10, Moscow, 117997
O. Goryainova
Institute of Gene Biology Russian Academy of Sciences
Email: liliya106@mail.ru
Ресей, ul. Vavilova 34/5, Moscow, 119334
A. Sachko
Institute of Gene Biology Russian Academy of Sciences
Email: liliya106@mail.ru
Ресей, ul. Vavilova 34/5, Moscow, 119334
T. Ivanova
Institute of Gene Biology Russian Academy of Sciences
Email: liliya106@mail.ru
Ресей, ul. Vavilova 34/5, Moscow, 119334
S. Tillib
Institute of Gene Biology Russian Academy of Sciences
Email: tillib@genebiology.ru
Ресей, ul. Vavilova 34/5, Moscow, 119334
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