Immunochemical detection specificity and molecular properties of Staphylococcus aureus-derived cytokine-like substances

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Abstract

Cytokines are main interaction regulators between cells of different origin in higher organisms and human. However similar molecules called cytokine-like substances (CLS) have been also found in more primitive organisms. Using immunochemical methods, CLS mimicking some human cytokines were detected in supernatants obtained after cultivation of different bacterial strains, including Staphylococcus aureus. This article was aimed at assessing immunochemical detection specificity and molecular properties of CLS derived from Staphylococcus aureus АТСС 25923 supernatants. Maximal CLS concentrations were detected using antibodies against human interleukin-1 receptor antagonist (IL-1rа), therefore justifying use of this cytokine for immunochemical specificity studies. According to the obtained data Staphylococcus aureus АТСС 25923 supernatants contained unspecific substance different from IL-1ra because it interacted not only with specific anti IL-1ra antibodies, but also with antibodies recognizing other proteins (for example, human enzyme superoxide dismutase). Upon this, Staphylococcus aureus АТСС 25923 supernatant had no competitive effect on interaction with immobilized IL-1rа, evidencing about the lack of specific antigen in the test material. For CLS identification they were purified from S. aureus АТСС 25923 supernatant using immunoaffinity chromatography on Sepharose column containing anti-human IL-1ra antibodies. In the eluate studied with SDS PAGE electrophoresis we have found heterogenous material characterized by four major bands with Mr = 50–60 kDa and 35 kDa. Importantly, it lacked 20 kDa signal specific for human IL-1ra. Each of four study electrophoretic components were extracted from the gel, digested with trypsin, and analyzed using mass-spectrometry that allowed to identify tryptic peptides highly homologous to that of staphylococcal protein A (SpA). Thus, the data obtained allowed to reveal SpA as a major eluate component. Highly likely it may be assumed that immunoglobulins used in enzyme immunoassay test kits for cytokine detection can react with secretory derivatives of staphylococcal immunoglobulin-binding proteins identified as CLS. Consequently, the so-called CLS detected by ELISA in supernatants derived after staphylococcus cultivation differ from true human cytokines, and their interaction with antibodies may be “non-specific”.

About the authors

N. P. Gorbunov

St. Petersburg Pasteur Institute; State Research Institute of Highly Pure Biopreparations

Email: amischenko1946@mail.ru

Junior Researcher, Head of Hybridoma Technology Laboratory

Russian Federation, St. Petersburg; St. Petersburg

Aleksandr M. Ischenko

St. Petersburg Pasteur Institute; State Research Institute of Highly Pure Biopreparations

Author for correspondence.
Email: amischenko1946@mail.ru

PhD (Biology), Head of Hybridoma Technology Laboratory

Russian Federation, St. Petersburg; St. Petersburg

A. G. Afinogenova

St. Petersburg Pasteur Institute

Email: amischenko1946@mail.ru

DSc (Biology), Leading Researcher, Head of Laboratory Testing Centre

Russian Federation, St. Petersburg

A. V. Zhakhov

St. Petersburg Pasteur Institute; State Research Institute of Highly Pure Biopreparations

Email: amischenko1946@mail.ru

Researcher, Hybridoma Technology Laboratory

Russian Federation, St. Petersburg; St. Petersburg

A. V. Trofimov

State Research Institute of Highly Pure Biopreparations

Email: amischenko1946@mail.ru

Head of the Research Group

Russian Federation, St. Petersburg

I. Ye. Eliseev

HSE University

Email: amischenko1946@mail.ru

PhD (Biology), Senior Researcher, School of Computer Science, Physics and Technology

Russian Federation, St. Petersburg

A. V. Zurochka

Institute of Immunology and Physiology UrB RAS

Email: amischenko1946@mail.ru

DSc (Medicine), Professor, Honored Scientist of the Russian Federation, Leading Researcher, Laboratory of Immunopathophysiology

Russian Federation, Ekaterinburg

L. O. Fomina

Institute of Immunology and Physiology UrB RAS

Email: amischenko1946@mail.ru

PhD Student, Laboratory of Immunopathophysiology

Russian Federation, Ekaterinburg

A. I. Fayzullina

Institute of Immunology and Physiology UrB RAS

Email: amischenko1946@mail.ru

PhD Student, Laboratory of Immunopathophysiology

Russian Federation, Ekaterinburg

V. A. Gritsenko

Institute for Cellular and Intracellular Symbiosis of the Ural Branch of the Russian Academy of Sciences, Orenburg Federal Research Center of the UrB RAS

Email: amischenko1946@mail.ru

DSc (Medicine), Professor, Scientific Secretary, Head Researcher

Russian Federation, Orenburg

A. S. Simbirtsev

St. Petersburg Pasteur Institute; State Research Institute of Highly Pure Biopreparations

Email: amischenko1946@mail.ru

RAS Corresponding Member, DSc (Medicine), Professor, Leading Researcher of Molecular Immunology Laboratory, Head Researcher

Russian Federation, St. Petersburg; St. Petersburg

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Copyright (c) 2025 Gorbunov N.P., Ischenko A.M., Afinogenova A.G., Zhakhov A.V., Trofimov A.V., Eliseev I.Y., Zurochka A.V., Fomina L.O., Fayzullina A.I., Gritsenko V.A., Simbirtsev A.S.

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