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

  • Rohini Krishna Kota
  • Krupanidhi Srirama
  • Prakash Narayana ReddyEmail author
Original Article


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.


Staphylococcus 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.

Informed consent



  1. Adhikari RP, Haudenschild C, Sterba PM, Sahandi S, Enterlein S, Holtsberg FW, Aman MJ (2016) Development of a novel multiplex electrochemiluminescent-based immunoassay for quantification of human serum IgG against 10 Staphylococcus aureus toxins. J Immunol Methods 430:33–42. CrossRefPubMedGoogle Scholar
  2. Atkins KL, Burman JD, Chamberlain ES, Cooper JE, Poutrel B, Bagby S, Jenkins ATA, Feil EJ, van den Elsen JM (2008) S. aureus IgG-binding proteins SpA and Sbi: host specificity and mechanisms of immune complex formation. Mol Immunol 45(6):1600–1611. CrossRefPubMedGoogle Scholar
  3. Babu L, Uppalapati SR, Sripathy MH, Reddy PN (2017) Evaluation of recombinant multi-epitope outer membrane protein-based Klebsiella pneumoniae subunit vaccine in mouse model. Front Microbiol 8:1805. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Burman JD, Leung E, Atkins KL, O’Seaghdha MN, Lango L, Bernadó P, Bagby S, Svergun DL, Foster TJ, Isenman DE, van den Elsen JM (2008) Interaction of human complement with Sbi, a staphylococcal immunoglobulin-binding protein indications of a novel mechanism of complement evasion by Staphylococcus aureus. J Biol Chem 283(25):17579–17593. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Clark EA, Crennell S, Upadhyay A, Zozulyab AV, Mackaya JD, Svergun DI, Bagby S, van den Elsen JMH (2011) A structural basis for staphylococcal complement subversion: X-ray structure of the complement-binding domain of Staphylococcus aureus protein Sbi in complex with ligand C3d. Mol Immunol 48:452–462. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Hong X, Qin J, Li T, Dai Y, Wang Y, Liu Q, He L, Lu H, Gao Q, Lin Y, Li M (2016) Staphylococcal protein a promotes colonization and immune evasion of the epidemic healthcare-associated MRSA ST239. Front Microbiol 7:951. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Mudili V, Makam SS, Sundararaj N, Siddaiah C, Gupta VK, Rao PVL (2015) A novel IgY-Aptamer hybrid system for cost-effective detection of SEB and its evaluation on food and clinical samples. Sci Rep 5:15151.
  8. Oliveira Calsolari RAD, Pereira VC, Araujo Junior JP, de Souza da Cunha MD, Ribeiro L (2011) Determination of toxigenic capacity by reverse transcription polymerase chain reaction in coagulase-negative staphylococci and Staphylococcus aureus isolated from newborns in Brazil. Microbiol Immunol 55(6):394–407. CrossRefPubMedGoogle Scholar
  9. Pauly D, Chacana PA, Calzado EG, Brembs B, Schade R (2011) IgY technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation. J Vis Exp 51:e3084. CrossRefGoogle Scholar
  10. Pierce IgY extraction kit (2017a) Product number 44918. (accessed on 10 July, 2018)
  11. Pierce IgY extraction kit (2017b) Product number 44918. (accessed on 10 July, 2018)
  12. Reddy PN, Ramlal S, Sripathy HM, Batra HV (2012) A simple and universal ligation mediated fusion of genes based on hetero-staggered PCR for generating immunodominant chimeric proteins. Gene 509:104–109. CrossRefPubMedGoogle Scholar
  13. Reddy PN, Shekar A, Kingston JJ, Sripathy HM, Batra HV (2013) Evaluations of IgY capture ELISA for sensitive detection of alpha hemolysin of Staphylococcus aureus without staphylococcal protein A interference. J Immunol Methods 391:31–38. CrossRefPubMedGoogle Scholar
  14. Reddy PN, Ramlal S, Sripathy HM, Batra HV (2014) Development and evaluation of IgY immunocapture ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference. J Immunol Methods 408:114–122. CrossRefPubMedGoogle Scholar
  15. Reddy PN, Nagaraj S, Sripathy MH, Batra HV (2015) Use of biotin-labeled IgY overcomes protein A interference in immunoassays involving Staphylococcus aureus antigens. Ann Microbiol 65(4):1915–1922. CrossRefGoogle Scholar
  16. Reddy PN, Srirama K, Dirisala VR (2017) An update on clinical burden, diagnostic tools, and therapeutic options of Staphylococcus aureus. Infect Dis 10:1179916117703999. CrossRefGoogle Scholar
  17. Sasso EH, Silverman GJ, Mannik M (1991) Human IgA and IgG F (ab′) 2 that bind to staphylococcal protein a belong to the VHIII subgroup. J Immunol 147(6):1877–1883PubMedGoogle Scholar
  18. Soleimanzadeh B, Amoozandeh A, Shoferpour M, Yolmeh M (2018) New approaches to modeling Staphylococcus aureus inactivation by ultrasound. Ann Microbiol 68(6):313–319CrossRefGoogle Scholar
  19. Upadhyay A, Burman JD, Clark EA, Leung E, Isenman DE, van den Elsen JM, Bagby S (2008) Structure-function analysis of the C3 binding region of Staphylococcus aureus immune subversion protein Sbi. J Biol Chem 283(32):22113–22120. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Wang W, Baloch Z, Jiang T, Zhang C, Peng Z, Li F, Fanning S, Ma A, Xu J (2017) Enterotoxigenicity and antimicrobial resistance of Staphylococcus aureus isolated from retail food in China. Front Microbiol 8:2256. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Yi SQ, Zhang XY, Yang YL, Yang Y, Liu SL, Fu L, Yu C, Chen W (2012) Immunity induced by Staphylococcus aureus surface protein a was protective against lethal challenge of Staphylococcus aureus in BALB/c mice. Microbiol Immunol 56(6):406–410. CrossRefPubMedGoogle Scholar
  22. Zhang L, Jacobsson K, Vasi J, Lindberg M, Frykberg L (1998) A second IgG-binding protein in Staphylococcus aureus. Microbiol 144(4):985–991. CrossRefGoogle Scholar

Copyright information

© Università degli studi di Milano 2019

Authors and Affiliations

  1. 1.Department of BiotechnologyVignan’s Foundation for Science, Technology and Research (VFSTR)Guntur DistrictIndia

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