SPR investigations of the formation of intermediate layer of the immunosensor bioselective element based on the recombinant Staphylococcal protein A

Abstract

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 nonspecifi c 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 K d value for SPA-Cys/IgG binding 8.5 ± 0.7× 10 -8 M (K a = 1.2 ± 0.1× 10 7 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 confi rms the effi ciency of SPA-Cys as an intermediate component for the creation of the immunosensor bioselective elements. K e y w o r d s: immunoglobulin, recombinant Staphylococcal protein A, surface plasmon resonance, protein immobilization, immunosensor, equilibrium binding constant.

Surface Plasmon Resonance Investigations of Bioselective Element Based on the Recombinant Protein A for Immunoglobulin Detection

“…The dependence of the immobilization level on the concentration of SPA-Cys was investigated. A linear part of this dependence was observed in the range from 0 to 0.5 μM SPA-Cys, whereas 2 μM SPA-Cys corresponds to the close-to-saturation level of immobilization [ 26 ]. According to the conversion factor of the SPR response into the value of surface density of the immobilized protein [ 28 ], this value at 2 μM SPA-Cys was 1.1 ± 0.2 ng/mm 2 .…”

“…2 , violet). Even taking into consideration a bigger size of BSA molecules, the average value 41 nm 2 of the sensor surface per each immobilized protein molecule indicates on a quite large area of free sensor surface [ 26 ].

Fig.

…”

“…In contrast to albumins, milk proteins (solution of the skim milk powder) demonstrated a high efficiency when being applied for the blocking procedure (Fig. 2 , red) [ 26 ]. However, after few days of exploitation with regeneration of the sensor surface, the injections of, for example, 40 μg/ml IgG resulted in the SPR response, which was much larger than the usual one.…”

“…To prevent nonspecific adsorption on the surface sites, which are left uncovered with immobilized proteins, the sensor surface was passivated with other proteins such as gelatin, milk proteins, BSA, or HSA. For this purpose, the consecutive injections of 120 μl of 0.2 mg/ml protein solution in PBS into the measuring cell were made followed by the incubation for 20 min and washing the surface with PBS until stabilization of the sensor signal [ 26 ].…”

Study on interactions of human IgG with immobilized anti-IgG or recombinant Staphylococcal protein A using surface plasmon resonance spectrometry

“…To prevent non-specific adsorption of studied components on the surface sites, which are left uncovered with the immobilized protein, a sensor surface was passivated with milk proteins. For this purpose, 120 µl of 200 µg/ml milk proteins in the working buffer solution (PBS) were injected into the measuring cell, then incubated at the pump switched off for 20 min, after that the surface was washed with PBS until stabilization of the sensor signal [11].…”