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Engineering of recombinant Staphylococcal protein A with cysteine residue (SPA-Cys) for preparation of affi nity chromatography stationary phase and formation of bioselective element of immunosensor. Methods. DNA sequences encoding IgG-binding region of SPA, His-tag and cysteine were genetically fused and expressed in E. coli. SPA-Cys was immobilized on maleimide-functionalized silica beads for affi nity chromatography stationary phase preparation and on a gold sensor surface as a bioselective element of immunosensor. Results. SPA-Cys was expressed at a high-level in a soluble form. The target protein was purifi ed and showed a high IgG-binding activity. The capacity of the obtained SPA-Cys-based affi nity chromatography stationary phase was 10-12 mg of IgG /ml. The purity of eluted IgG was more than 95 % in one-step purifi cation procedure. The developed SPA-Cys-based bioselective element of immunosensor selectively interacted with human IgG and did not interact with the control proteins. Conclusions. The recombinant Staphylococcal protein A with cysteine residue was successfully used for the preparation of affi nity chromatography stationary phase and formation of the bioselective element of immunosensor. K e y w o r d s: antibodies, recombinant Staphylococcal protein A, protein immobilization, affi nity chromatography, immunosensor, surface plasmon resonance.

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.

The developed surface plasmon resonance (SPR) biosensor based on the recombinant Staphylococcal protein A with an additional cysteine residue (SPA-Cys) used as a biorecognition component showed a good selectivity and sensitivity for the immunoglobulin detection. The developed biosensor with SPA-Cys-based bioselective element can also be used as a first step of immunosensor creation. The successful immobilization of SPA-Cys on the nanolayer gold sensor surface of the SPR spectrometer was performed. The efficiency of blocking nonspecific sorption sites on the sensor surface with milk proteins, gelatin, BSA, and HSA was studied, and a rather high efficiency of using gelatin was confirmed. The SPR biosensor selectively interacted with IgG and did not interact with the control proteins. The linear dependence of the sensor response on the IgG concentration in the range from 2 to 10 μg/ml was shown. Using the calibration curve, the IgG concentration was measured in the model samples. The determined concentrations are in good agreement (r 2 = 0.97) with the given concentration of IgG.

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Aim. The creation of genetically engineered fusion protein SPA-BAPmut and its application as a secondary immunoreagent in immunoassays. Methods. Gene cloning, PCR, electrophoresis, DNA sequencing, bacteria cells culturing, protein expression and purification, ELISA, Western-blotting were used. Results. The DNA sequences encoding Staphylococcus aureus protein A (SPA) and bacterial alkaline phosphatase with enhanced catalytic activity (BAPmut) were used for construction of gene encoding fusion protein SPA-BAPmut that was expressed in the high-productive Escherichia coli system and obtained in a soluble form. Cultivation conditions to provide a high-level expression of SPA-ÂAPmut (> 1 g/l) were determined. The target protein was obtained with purity more than 95 % using ²ÌÀÕ method. SPA-ÂAPmut is thermostable, and both parts of fusion protein (SPA and BAPmut) retain their IgG binding and alkaline phosphatase activity for a long time. SPA-BAPmut was used as a substitute of secondary an-tibodies in immunoassays. As little as 5 ng of the antigen could be detected in Western blotting and 1 mg/ml of IgG in ELISA. Conclusions. The possibility of using SPA-ÂAPmut as universal secondary immunoreagent for different types of immunoassays was shown.

Aim. The obtaining of bioaffinity sorbent based on the immobilized protein A of S. aureus (SPA) using two cellulose-binding domains (CBD), and its application for purification of antibodies. Methods. The DNA sequences encoding SPA and two CBD were genetically fused, expressed in the high-productive Escherichia coli system and the protein SPA-CBD2 was obtained in a soluble form. The SPA-CBD2 fusion protein was affinity immobilized on the microcrystalline cellulose. Results. Capacity of bioaffinity sorbent (1 mg SPA-CBD2/1 ml CC31-cellulose), dynamic capacity (3 mg mouse IgG/1 ml bioaffinity sorbent), efficiency and stability during prolonged storage were determined. The bioffinity sorbent was used for purification of antibodies. The purity of antibodies in eluted fractions was more than 95 %. The purified antibodies detected target antigens with a high sensitivity. Conclusions. The designed bioaffinity sorbent provides obtaining pure poly- and monoclonal antibodies in functionally active form and can be useful for the fractionation of mouse immunoglobulin G