IMMUNOCHEMICAL DETECTION SPECIFICITY AND MOLECULAR PROPERTIES OF STAPHYLOCOCCUS AUREUS-DERIVED CYTOKINE-LIKE SUBSTANCES

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”.