Biopolym. Cell. 2015; 31(4):301-308.
http://dx.doi.org/10.7124/bc.0008EF
Molecular and Cell Biotechnologies
SPR investigations of the formation of intermediate layer of the immunosensor bioselective element based on the recombinant Staphylococcal protein A
1Rachkov A. E., 1, 2Bakhmachuk A. O., 1Gorbatiuk O. B., 1, 3Matsishin M. J., 4Khristosenko R. V., 4Ushenin Iu. V., 1, 3Soldatkin A. P.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine,
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. Educational and Scientific Center "Institute of Biology",
    Taras Shevchenko National University of Kyiv
    64/13, Volodymyrska Str., Kyiv, Ukraine, 01601
  3. Institute of High Technologies,
    Taras Shevchenko National University of Kyiv
    2, korp.5, Pr. Akademika Hlushkova, Kyiv, Ukraine, 03022
  4. V. Ye. Lashkaryov Institute of Semiconductor Physics, NAS of Ukraine
    41, Prospect Nauki, Kyiv, Ukraine, 03028

Abstract

Aim. To investigate the formation of an intermediate layer of the immunosensor bioselective element based on the recombinant protein A from Staphylococcus aureus with cysteine residue (SPA-Cys) and its interactions with human IgG using the SPR spectrometer «Plasmon». Methods. The activity of the immune components applied was tested by ELISA. The spectrometry of surface plasmon resonance was used for studying protein immobilization on a gold sensor surface and interactions between the immobilized SPA-Cys and human immunoglobulin. Results. A direct dependence of the sensor response on the concentration of SPA-Cys in the range of 0.2 to 2 µM at its immobilization was demonstrated. The efficiency of blocking nonspecific adsorption sites on the sensor surface with milk proteins and the direct dependence of the sensor response on IgG concentration and surface density of immobilized SPA-Cys were shown. Fitting the experimental data to a Langmuir plot yields a Kd value for SPA-Cys/IgG binding 8.5 ± 0.7× 10-8 M (Ka = 1.2 ± 0.1× 107 M–1). The determined equilibrium binding constant indicates a quite strong interaction and its value is consistent with the literature data. Conclusions. A successful immobilization of SPA-Cys on a gold surface of the SPR spectrometer while preserving its high immunoglobulin-binding activity, selectivity and stability of the sensor response confirms the efficiency of SPA-Cys as an intermediate component for the creation of the immunosensor bioselective elements.
Keywords: immunoglobulin, recombinant Staphylococcal protein A, surface plasmon resonance, protein immobilization, immunosensor, equilibrium binding constant.
Full text: (PDF, in English)

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