IgY antibodies of chicken do not bind staphylococcal binder of immunoglobulin (Sbi) from Staphylococcus aureus
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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.
KeywordsStaphylococcus aureus Immunoglobulin Y (IgY) Immunoglobulin G (IgG) Staphylococcal binder of immunoglobulin (Sbi) Staphylococcal protein A (SpA) False positives ELISA
Prakash Narayana Reddy is thankful to INSPIRE division of Department of Science and Technology, Govt. of India for supporting with fellowship and research grant. Authors are thankful to management and administration of Vignan’s Foundation for Science, Technology and Research (VFSTR) for providing infrastructural facilities and necessary support. Rohini Krishna Kota is a research scholar and teaching assistant supported by VFSTR.
Prakash Narayana Reddy is an INSPIRE Faculty awardee supported by the Department of Science and Technology (DST), Govt. of India (INSPIRE Faculty award: DST/INSPIRE/04/2017/000565). This study was supported from INSPIRE Faculty research grant from Department of Science and Technology (DST), Govt. of India.
Compliance with ethical standards
Conflicts of interest
Authors declare no conflicts of interest.
Research involving human participants and/or animals
Chicken and mice were maintained under hygienic conditions in animal housing facility of Vignan’s pharmacy college which is adjacent to VFSTR. Necessary permissions for using animals were sought from Institutional Animal Ethical Committee (IAEC) of Vignan’s Pharmacy College, Vadlamudi, Andhra Pradesh. Mice were provided with protein rich pellet feed and mineral water. Chicken were provided with commercial poultry feed used in local poultry farms. Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines were followed for care and use of animals and the Institutional Animal Ethical Committee (IAEC) of Vignan’s Pharmacy College approved the protocols and procedures employed in this study.
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