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

Abstract

Objective: Transferrin (Tf) exists in two forms in blood plasma: iron-containing holo-Tf and iron-free apo-Tf forms. An important biochemical marker of diseases associated with iron deficiency or excess is the quantitative ratio of these forms in human blood plasma. Methods: Application of the fluorescence polarization immunoassay (FPIA) method and the use of recombinant camel nanobodies as a recognition reagent for the rapid determination of holo-Tf and apo-Tf will allow the development of a rapid method for determining two transferrin conformations. Results and Discussion: Conjugates of camel nanobodies aTf1 and aTf2 to holo- and apo-forms of human transferrin (Tf) with fluorescein isothiocyanate (FITC) were synthesized and characterized. Concentrations of FITC-aTf1 and FITC-aTf2 conjugates (2.5–5 nM) with an optimal signal-to-noise ratio were selected and the binding kinetics of the resulting FITC-aTf1 and FITC-aTf2 conjugates to holo- and apo-Tf was studied using the fluorescence polarization method. It was shown that complete binding of FITC-aTf1 and FITC-aTf2 conjugates to holo- and apo-Tf is 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 to be 30.7 ± 0.3 and 15.3 ± 0.2 nM, respectively. It was demonstrated that incubation of FITC-aTf1 and FITC-aTf2 conjugates with other human proteins—lactoferrin, serum albumin and lysozyme did not change the fluorescence polarization signal, indicating high specificity of the assay. It was shown that the FITC-aTf1/apo-Tf and FITC-aTf2/holo-Tf reagent pairs also did not exhibit binding to each other, confirming the affinity of FITC-aTf1 and FITC-aTf2 conjugates to holo- and apo-Tf, respectively. Conclusions: 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.