IgY antibodies of chicken do not bind staphylococcal binder of immunoglobulin (Sbi) from Staphylococcus aureus

• Published in: Annals of microbiology 2019-05, Vol.69 (5), p.531-540
• Main Authors: Kota, Rohini Krishna, Srirama, Krupanidhi, Reddy, Prakash Narayana
• Format: Article
• Language: English
• Subjects: Affinity; Analysis; Antigen-antibody reactions; Antigens; Applied Microbiology; Binding proteins; Biomedical and Life Sciences; Biotin; Cloning; Enzyme-linked immunosorbent assay; Escherichia coli; Exotoxins; Genetic aspects; Immune response; Immunoassay; Immunogenicity; Immunoglobulin G; Immunoglobulins; Life Sciences; Medical Microbiology; Microbial Ecology; Microbial Genetics and Genomics; Microbiology; Mycology; Original Article; Protein A; Protein binding; Proteins; SbI protein; Staphylococcus aureus; Streptavidin; Western blotting
• ISSN: 1590-4261; 1869-2044
• DOI: 10.1007/s13213-019-1441-8

The immunoglobulin (Ig) binding proteins of Staphylococcus aureus namely staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulin (Sbi) are responsible for false positives during immunoassays. Avian IgY antibodies were reported to have no affinity to SpA and thus are safe for use in immunoassays. However, the behaviour of Sbi with IgY was not reported. The purpose of the present study is to evaluate the interactions between IgY antibodies and Sbi protein from different S. aureus strains. Initially, heterologous cloning and expression of complete sbi gene in Escherichia coli was undertaken. Recombinant Sbi protein was utilized to generate polyclonal anti-Sbi IgY and anti-Sbi antibodies in chicken and BALB/c mice respectively. Indirect ELISA and Western blotting were performed to evaluate the reactivity of anti-Sbi antibodies. Non-reducing PAGE followed by Western blotting and double-antibody sandwich dot-ELISA were performed to analyze the reactivity of IgY antibodies with recombinant Sbi and native Sbi from S. aureus strains. To avoid the possible interference of enzyme-conjugated secondary antibodies from mammalian sources, mouse anti-Sbi revealing antibodies were labeled with biotin so that streptavidin-HRP was used as developing reagent for chromogenic reaction. Sbi was highly immunogenic in chicken and mouse with antibody titers of 1:128,000 and 1:64,000 dilutions respectively. We observed that unimmunized IgY antibodies showed no affinity to either recombinant Sbi or native Sbi from S. aureus strains in Western blotting and double antibody sandwich ELISA. In view of these observations, we recommend that IgY antibodies are safe and free from false positives due to SpA and Sbi in immunoassays involving detection of S. aureus antigens/exotoxins.