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

Мұқаба

Дәйексөз келтіру

Толық мәтін

Ашық рұқсат Ашық рұқсат
Рұқсат жабық Рұқсат берілді
Рұқсат жабық Тек жазылушылар үшін

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

Рұқсат жабық

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, 119234

S. Eremin

Lomonosov Moscow State University

Email: liliya106@mail.ru

Department of Chemistry

Ресей, Leninskie gory 1/3, Moscow, 119234

I. 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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1. JATS XML
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.

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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.

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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.

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5. Fig. 4. Specificity of FIT CaT f1 and FITC-aTf2 nanobodies in interaction with other proteins; pH 7.4, 25°C.

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

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