Solid-phase capture of proteins, spores, and bacteria

Current methods for the detection of pathogens in food and water samples ge nerally require a preenrichment step that allows selective enrichment of th e test organism. The objective of this research was to eliminate an enrichm ent step to allow detection of bacteria directly in food and water samples in 30 min. A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed. This fo rmat, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when t esting food samples. Detection of bound targets was done using existing enz yme-linked immunosorbent assay (ELISA) protocols. Four antibodies (anti- Es cherichia coli O157:H7, -Bacillus globigii, -bovine serum albumin [BSA], an d -ovalbumin [OVA]) were covalently coupled to various glass and ceramic be ads. Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 <mu>g) were dete cted. Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-B. globigii and an ti-E. coli O157:H7 modified beads. The lower limit of detection for both E. coli O157:H7 and B. globigii was 1 spore/cell independent of the sample si ze. The activity of anti-B. globigii modified beads declined after 3 days. Anti-E. coli O157:H7 modified beads declined in the capture ability after 2 days in various storage buffers. Storage temperature (4 and 25 degreesC) d id not influence the stability. The ImmunoFlow technology is capable of cap turing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.