Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products.

Staphylococcal enterotoxins (SEs), produced by Staphylococcus aureus, are a major cause of staphylococcal food poisoning. Traditionally, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse passive latex agglutination with rabbit antibody IgG have been used to detect SEs. However, most of these kits require a long processing time and there is a risk of false-positive results since IgG reacts nonspecifically with protein A produced by S. aureus. In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2 ng/ml within 15 min in milk. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1 ng/ml in milk; the SEs were detected within 12 min and specialized skills were not required. The ELISA and LFD detected SEA in dairy products artificially contaminated with S. aureus, including ice cream, yogurt, and café au lait, in a dose-dependent manner. In conclusion, IgY allows highly specific detection of SEs, and ELISAs, LFDs, and immunopillar chips should be useful tools for screening SEs in milk and dairy products.

Immuno-pillar chip: a new platform for rapid and easy-to-use immunoassay.

We present a new rapid and easy-to-use immunoassay chip which we have named the immuno-pillar chip. It has hydrogel pillars, fabricated inside a microchannel, with many antibody molecules immobilized onto 1 µm diameter polystyrene beads. To evaluate the chip performance, we applied it to the sandwich assay of C-reactive protein (CRP), α-fetoprotein (AFP) and prostate-specific antigen (PSA), a cardiac and inflammation marker, tumors and prostate cancer markers, respectively. For detection of disease markers, we confirmed the chip provides rapid analysis (total assay time of about 4 min) with high sensitivity, it is easy-to-use (no special skills are needed), and it uses small volumes of the sample and reagent (0.25 µL each). Moreover, multiplex assay for three biomarkers was also possible. Additionally, the immuno-pillar chip has a big advantage of having hardly any influence on the assay results even if the introduction quantities of the sample or reagents are different.