Development of the test system based on enzyme immunoassay for the detection of recombinant <i>Streptococcus</i> pneumoniae pneumolysin

Abstract

Backgraund. Pneumolysin (Ply) is a hemolytic toxin of Streptococcus pneumoniae (S. pneumoniae) expressed by all strains of pneumococci. The use of sandwich enzyme-linked immunosorbent assay (ELISA) can be a simple, fast and effective way of its qualitative and quantitative determination in biological fluids.Aim. To develop and evaluate the specificity of sandwich ELISA test system for qualitative and quantitative determination of recombinant Ply (rPly) of S. pneumoniae.Materials and methods. Immobilized on the solid phase rabbit’s polyclonal antibodies (pAbs) to rPly were used as recognition antibodies in sandwich ELISA. The studied antigens were added to the pAbs (rPly). The reaction was manifested by using detecting mouse monoclonal IgG1 (rPly) – antibodies conjugated with horseradish root peroxidase. The specificity of the test system was evaluated when using recombinant α-hemolysin (rα-Hly) and water-soluble S. aureus antigens as reference preparations.Results. Using sandwich ELISA, rPly was detected at a concentration of 0.15 µ / ml. The test system was characterized by specificity, which was confirmed by the absence of reaction with recombinant rα-Hly of Staphylococcus aureus (S. aureus). Reference preparations of water-soluble surface antigens of S. aureus strains No 209, 1986,1991 and Cowan I gave a false positive reaction due to the presence of protein A (SpA) in their composition, a thermostable surface protein expressed by many strains of staphylococci capable of binding immunoglobulins via Fc-fragment or Fab fragments of the V3H domain of the B cells receptor. A negative reaction was obtained with antigens from the S. aureus wood 46 strain, which does not have the spa gene encoding SpA expression. The presence of protein A in preparations of water-soluble S. aureus antigens was confirmed in the ELISA inhibition assay.Conclusion. Sandwich ELISA has been developed for qualitative and quantitative determination of S. pneumoniae Ply. The conducted studies have confirmed the specificity of the test system.