Use of biotin-labeled IgY overcomes protein A interference in immunoassays involving Staphylococcus aureus antigens

Abstract

Staphylococcal protein A (SpA) is an immunoglobulin-binding protein secreted by all Staphylococcus aureus strains and is responsible for high false positives during immunoassays. Avian IgY were reported to eliminate SpA interference when used as a capture antibody in a double antibody sandwich format. But this procedure requires generating two antibodies in different animals for capture and revealing antibodies. In the present study, a simple enzymelinked immunosorbent assay (ELISA) was developed and evaluated for detection of staphylococcal enterotoxin B without any interference by SpA. Biotin-labeled IgY were used for probing for staphylococcal enterotoxin B (SEB) so that streptavidin-horseradish peroxidase (HRP) could be used for color development instead of anti-chicken IgG-HRP, which interferes in the assay by binding to SpA. Western blotting, dot ELISA, and polymerase chain reaction (PCR) were performed to validate the results of the biotin IgY ELISA to show that the assay was free from SpA interference. Large numbers of S. aureus strains were screened for SEB production. Sensitivity of the assay was~10 ng/ml of SEB in spiked milk samples. The ELISA was specific to SEB, with no crossreactivity to other closely related enterotoxins (SEA, SEC, SED, SEE). The presently described ELISA is highly effective in eliminating SpA interference and this method does not require the need for two different antibodies, as in the sandwich ELISA.

RETRACTED ARTICLE: A novel IgY-Aptamer hybrid system for cost-effective detection of SEB and its evaluation on food and clinical samples

“…Due to the utility of two different bio-ligands raised against same target molecule, sandwich immunoassays play a crucial role in sensitive and selective detection of target toxic agents like SEB. In development of sandwich immunoassays for detection of SEB, utility of IgY is great advantage, since IgY is free from interaction with protein A, which is major obstacle in development of SEB detection systems 26 . Moreover, ease of production in bulk quantities, moderate sensitivities and high specificities together with free from animal ethical committee issues makes it significance to be a better alternative over monoclonal as well as polyclonal antibodies.…”

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

“…In our earlier work, we reported the efficacy of immunoglobulin Y over immunoglobulin G in eliminating false positives due to SpA during detection of S. aureus exotoxins (Reddy et al 2013). Efficacy of IgY antibodies over IgG was demonstrated by IgY capture ELISA (Reddy et al 2013) and IgY immunocapture PCR (IC-PCR) (Reddy et al 2014) in double antibody sandwich ELISAs and by using biotin labeled IgY in single antibody assay (Reddy et al 2015). Additionally, a hybrid Aptamer Linked ImmunoSorbent Assay (ALISA) procedure was developed using IgY capture antibody and anti-staphylococcal enterotoxin B (SEB) aptamer for detection of SEB (Mudili et al 2015).…”

“…Avian antibodies from chickens (IgY) are increasingly used for developing diagnostic procedures due to low costs and higher yields than from their mammalian counterparts (Reddy et al 2013(Reddy et al , 2014. Antibodies can be harvested from egg yolks without bleeding or using any invasive techniques (Reddy et al 2015). Heavy chains of IgY are composed of four constant domains unlike three constant domains of mammalian IgG.…”

Immunoglobulin Y for Potential Diagnostic and Therapeutic Applications in Infectious Diseases

“…IgY has been employed as a method to detect the bacterium, as well as its associated proteins and toxins to prevent S. aureus-induced food poisoning. IgY against S. aureus or its endotoxins has been used in sandwich ELISAs, immuncapture polymerase chain reaction ELISAs, lateral flow devices, immunopillar chips, immunochromatography, and fluorescence resonance energy transfer assays (245)(246)(247)(248)(249)(250)(251)(252). The success of these approaches is considered due to IgY's lack of reactivity with protein A of S.aureus, which often results in false positives results in other assays (253).…”