Development of an anchor bispecific nanoantibody to improve the efficiency of antigen immobilization and detection in a well of a polystyrene plate

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Abstract

Immunoassay (IA) methods performed in the wells of a polystyrene microplate are the basis of diagnostic studies. In the “sandwich” IA, a fundamentally important initial stage is the immobilization of anchor antibodies in the well of the plate, designed for specific binding of a given antigen from a biological fluid. One of the very promising options for antigen-recognizing molecules are single-domain antibodies (nanoantibodies, Nb). The use of Nbs as anchor antibodies is hampered by their low efficiency of functioning after passive adsorption in the well of the plate. The development of a new format and immobilization method in the case of NT are fundamentally important for overcoming this problem. This work describes the development of a new format of an anchor bispecific nanoantibody (anchor-Nb) to improve the efficiency of both passive adsorption of anchor--NT and subsequent stages of immobilization and detection of the target antigen in the well of a polystyrene plate.

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About the authors

S. V. Tillib

Institute of Gene Biology, Russian Academy of Sciences

Author for correspondence.
Email: sergei.tillib@gmail.com
Russian Federation, Moscow

M. V. Panasyuk

Institute of Gene Biology, Russian Academy of Sciences

Email: sergei.tillib@gmail.com
Russian Federation, Moscow

O. S. Goryainova

Institute of Gene Biology, Russian Academy of Sciences

Email: sergei.tillib@gmail.com
Russian Federation, Moscow

T. I. Ivanova

Institute of Gene Biology, Russian Academy of Sciences

Email: sergei.tillib@gmail.com
Russian Federation, Moscow

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2. Fig. 1. Properties of two single-domain antibodies (yak-5 and yak-7) that bind polystyrene with high affinity, selected by phage display. The upper part of the figure shows the averaged results of the accelerated enzyme immunoassay of binding to empty (without preliminary blocking) and 1% BSA-blocked wells of a polystyrene plate (MaxiSorp, Nunc) of two new “sticky” NTs, yak-5 and yak-7, as well as the previously obtained control NT against human lactoferrin (aLF-6). Wells without NTs also served as a control. The vertical bars show the scatter areas of data from three parallel experiments. Table 1 shows some properties of the selected NTs and the amino acid sequences of their hypervariable regions.

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3. Fig. 2. Creation and use of the novel anchor bispecific nanoantibody LF6-L1-yak7 for antigen (hLF) detection. (A) Scheme of the expression construct used for the synthesis of bispecific nanobodies. From left to right, the construct shows: lactose promoter region (Plac/operator); ribosome binding site (RBS); transcription start (arrow); signal peptide for periplasmic localization (pelB); sequences encoding nanobodies (aLF-6 and yak-7) separated by the linker sequence L1 [9]; HA tag sequences (YPYDVPDYA) and six histidine residues at the very end. (B) – Electrophoretic separation of affinity purified nanobodies in 5-19% gradient SDS-polyacrylamide gel. The arrow shows the position of the bispecific nanobodies (bs-NT) and the purified monomeric nanobody aLF-6 (NT) is applied for comparison. (B) Calibration curve for human lactoferrin (hLF) detection constructed from averaged colony count data obtained using the developed sandwich immunoassay with the anchor bispecific nanobody (LF6-L1-yak7) and the detection nanobody aLF-5 exposed on the surface of the recombinant bacteriophage M13. Black circles indicate averaged data obtained for different hLF concentrations (0, 0.5, 1, 2, 4, 8 and 16 ng/mL). The asterisk indicates the average data for the determination of hLF in a sample of diluted human blood plasma (the black asterisk corresponds to data on a noticeable increase in the detectable hLF as a result of pre-treatment of the plasma in order to dissociate possible complexes of the detectable antigen with other components).

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