Development of a recombinant antibody for the detection of activated MBL

Staphylococcal protein A (Spa) secreted by all Staphylococcus aureus strains is the major hindrance in development of specific immunoassays for detecting S. aureus antigens, because of its characteristic feature of binding to Fc region of most mammalian immunoglobulins and also to Fab region of certain classes of mammalian immunoglobulins. Immunoglobulin Y (IgY) is the avian equivalent of mammalian IgG which does not have any affinity to Spa. In the present study we report that using chicken egg yolk IgY over mammalian IgG as capture antibody prevents both soluble and surface bound protein A from causing false positives quantified by chicken anti-protein A antibodies. This was demonstrated by development of sandwich ELISA for detection of alpha hemolysin toxin from culture supernatants of S. aureus strains with anti alpha hemolysin IgY as capture and rabbit anti alpha hemolysin IgG as revealing antibody. This indirect sandwich ELISA was evaluated onto a large number of S. aureus isolates recovered from clinical sources for alpha hemolysin secretion. Results of sandwich ELISA were compared with PCR and Western blot analysis. The immunoassay is highly specific and has high sensitivity of detecting less than 1 ng/ml. This procedure is highly effective in eliminating Spa interference and can be extended to detection of other important superantigen toxins of S. aureus.