RETRACTED ARTICLE: A novel IgY-Aptamer hybrid system for cost-effective detection of SEB and its evaluation on food and clinical samples

Abstract

In the present study, we introduce a novel hybrid sandwich-ALISA employing chicken IgY and ssDNA aptamers for the detection of staphylococcal enterotoxin B (SEB). Cloning, expression and purification of the full length recombinant SEB was carried out. Anti-SEB IgY antibodies generated by immunizing white leg-horn chickens with purified recombinant SEB protein and were purified from the immunized egg yolk. Simultaneously, ssDNA aptamers specific to the toxin were prepared by SELEX method on microtiter well plates. The sensitivity levels of both probe molecules i.e., IgY and ssDNA aptamers were evaluated. We observed that the aptamer at 250 ngmL−1 concentration could detect the target antigen at 50 ngmL−1 and the IgY antibodies at 250 ngmL−1, could able to detect 100 ngmL−1 antigen. We further combined both the probes to prepare a hybrid sandwich aptamer linked immune sorbent assay (ALISA) wherein the IgY as capturing molecule and biotinylated aptamer as revealing probe. Limit of detection (LOD) for the developed method was determined as 50 ngmL−1. Further, developed method was evaluated with artificially SEB spiked milk and natural samples and obtained results were validated with PCR. In conclusion, developed ALISA method may provide cost-effective and robust detection of SEB from food and environmental samples.

Highly Sensitive Colorimetric Biosensor for Staphylococcal Enterotoxin B by a Label-Free Aptamer and Gold Nanoparticles

“…Staphylococcus aureus is a unique and notorious pathogen due to its wide range of virulence factor such as α -hemolysin, several enterotoxins (as many as 23, ranging from SEA to SHV), TSST and other secretary proteins (Balaban and Rasooly, 2000 ; Singh et al, 2010 ). Among the virulence, the heat-stable staphylococcal enterotoxins B (SEB) are the most effective cause of food-borne illness, as it cause infection nanogram levels, can cross gastro-intestinal barrier and easy to aerosolize which implies to use as potent biological weapon (Balaban and Rasooly, 2000 ; Mudili et al, 2015 ). Moreover, heat stable SEB can remain unaffected in food after cooking and even heat treatment which only inactivates or kills S. aureus cells.…”

“…In our study we have employed new generation affinity molecule aptamer as recognition probe which is advantageous in comparison to available ligands in term of specificity, sensitivity and stability (Mondal et al, 2015 ; Lavu et al, 2016 ). Moreover it is known fact mammalian antibodies have affinity toward Protein A (SpA) which always leads to non-specific and false positive results limits their application of immunoassays (Reddy et al, 2014 ; Mudili et al, 2015 ). Conventional nucleic acid based need additional steps of sample purification and extraction of DNA and the sensitivity will be easily compromised by interfering matrix associated inhibitory substances (Mudili et al, 2015 ).…”

Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review

“…Another ELISA test using IgY against the staphylococcal enterotoxin B (SEB) of S. aureus as capture antibody and specific ssDNA aptamers coupled to biotin as revealing was developed by Mulidi et al This assay was efficient in detecting SEB, but also reacted with others staphylococcal toxins, such as staphylococcal endotoxins A (SEA) and C (SEC), toxic shock syndrome toxin (TSST) and α-hemolysin [111].…”

Current advances in aptamer-assisted technologies for detecting bacterial and fungal toxins

“…Other systems have introduced a novel hybrid sandwich-ALISA via employing chicken IgY (a type of antibody found in birds, but not in mammals) associated ssDNA aptamers selected by the SELEX method for detecting this toxin (Mudili et al 2015). A single aptamer at the concentration of 250 ng ml À1 could identify the target at 50 ng ml À1 .…”

“…The limitation of this method was determining 50 ng ml À1 in spiked milk and natural specimens (Mudili et al 2015).…”