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.
Subject(s)
Aptamers, Nucleotide , Enterotoxins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulins/immunology , Staphylococcal Food Poisoning/diagnosis , Staphylococcal Infections/diagnosis , Animals , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/genetics , Chickens , Cloning, Molecular , Cost-Benefit Analysis , Enterotoxins/blood , Enterotoxins/genetics , Enzyme-Linked Immunosorbent Assay/economics , Gene Expression , Humans , Nucleic Acid Conformation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SELEX Aptamer Technique , Sensitivity and SpecificityABSTRACT
An outbreak of viral encephalitis occurred in Gorakhpur, India, from July through November 2005. The etiologic agent was confirmed to be Japanese encephalitis virus by analyzing 326 acute-phase clinical specimens for virus-specific antibodies and viral RNA and by virus isolation. Phylogenetic analysis showed that these isolates belonged to genogroup 3.
