Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cells

Abstract

Aims: To develop methods to assess the efficiency of immunomagnetic separation (IMS). Methods and Results: The capturing efficiency of biosorbents for Escherichia coli O157:H7, constructed using streptavidin-coated magnetic beads and biotinylated antibodies, was tested using both in vivo and ATP bioluminescence. Both methods were suitable for the enumeration of bacteria captured by the biosorbents. The level of both ATP and in vivo bioluminescence depended on the media used, but was unaffected by the magnetic beads. The capture efficiency depended on time and sample volume, but did not depend on the length of spacer arm of the biotinylation agent. For cell concentrations of £ 10 5 cfu ml )1 , in a 1-ml sample volume, nearly 80-85% recovery of the pathogen was observed after 0AE5 h of incubation. For an 11-ml sample containing 10 4 cfu ml )1 , maximum recovery (50% of cells) was achieved only after 2 h incubation. Conclusions: The detection limit of an ATP-based bioluminescent assay for E. coli O157:H7 was reduced by 1 log cycle after optimization of IMS. The bioluminescent methods could be used for screening and testing the affinity of antibodies or other affinity elements of biosorbents towards live bacterial cells. Significance and Impact of the Study: Bioluminescent assays provide an easy way to optimize conditions for the capture of bacteria by biosorbents in real time.

Determination of microbial load for different beverages and foodstuff by assessment of intracellular ATP

“…The efficiency of bacterial capture by the biosorbent depends on the properties of the capturing element (antibody, bacteriophage, lectin, etc.) as well as on environmental conditions, such as pH, media composition, osmomolarity and water activity (Sun, Khosravi, Albrechtsen, Brovko, & Griffiths, 2002). The method has been used to detect food pathogens in various matrices, including water, ground beef, apple juice and milk (Hunter & Lim, 2010;Lee & Deininger 2004), increasing specificity and sensitivity of ATP measurement.…”

Dynabeads protein G antibody conjugates combined with modified brain heart infusion broth for the enrichment and separation of Bacillus cereus in artificially contaminated vegetables

“…be formed due to capture of only one cell by several particles, the calculation method based on the separated cells could be underestimated since the plating method only counts the colonies on the agar plates (27). The CE based on the counts of cells bound to the magnetic beads was less than that based on the unbound cells in the supernatant (19).…”

Simultaneous Detection of Escherichia coli O157:H7 and Salmonella Typhimurium: The Use of Magnetic Beads Conjugated with Multiple Capture Antibodies

“…Again, as discussed previously, the recovery of Salmonella from inoculated food samples was not as favorable as the recovery of E. coli. Sun et al (2002) showed that multiple bacterial cells could be captured by one immunomagnetic bead and that one cell could bind to multiple beads, thus creating clusters. Since one bead can bind multiple bacterial cells, we aimed to develop an IMB-based assay to capture multiple pathogens in a single step.…”