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.
Highlights
To evaluate the reliability and ease of access, developed method was evaluated onto several food and clinical samples originated from India were undertaken and results of the developed method showed very promising for detection of SEB in tested samples
The expression host E. coli BL21 DE3 colonies transformed with desired gene was recovered and sub cloned on to LB agar medium with suitable antibiotics as well as same was inoculated into LB broth
We further evaluated the same matrix with microtitre plate format onto various toxigenic and non toxigenic strains of S. aureus and other related bacteria and study results revealed that, developed method was highly specific to the SEB toxin alone (Fig. 6)
Summary
Sixty-eight methods based on antibodies and analytical instrumentation, including surface plasmon resonance[9], piezoelectric crystal immunosensing[10], magnetoelastic sensing[11], liquid chromatography mass spectrometry[12], surface-enhanced Raman scattering probe[13], cantilever sensing[14], electrochemical[15] and photonic crystal lab-on-a-chip methods[16] are already available for the detection of SEB These reported methods have their own limitations such as requirement of expertise personnel for data interpretation, sophisticated instrumentation, lengthy protocol times, and diseconomy. Aptamers have many advantages over antibodies, such as more stable, easier modification, easier synthesis, and higher affinity, and they can be fluorescently labeled and do not require experimental animals for synthesis[20,21] Due to these properties, a variety of aptamer-based analytical methods including electrochemistry, fluorescence, atomic force microscope, and quartz crystal microbalance have been developed for molecular recognition and detection of threat agents[22,23,24,25]. To evaluate the reliability and ease of access, developed method was evaluated onto several food and clinical samples originated from India were undertaken and results of the developed method showed very promising for detection of SEB in tested samples
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