Author
 |
Brewster, Jeffrey |
|
Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 4/29/2003 Publication Date: 10/1/2003 Citation: Brewster, J.D. 2003. Effect of bead diameter and flow rate in micro-immunoaffinity column capture of e. coli 0157:h7. Journal of Microbiological Methods. Interpretive Summary: Rapid, sensitive assays capable of detecting small numbers of pathogenic bacteria in foods are needed by regulatory inspection personnel and others in agriculture and food industries working to reduce foodborne illnesses, such as those caused by E. coli O157:H7. Current techniques are not able to detect very low levels of pathogens, so it is necessary to culture samples for a period of time to allow the pathogen to grow and increase in concentration before detection. This enrichment process adds hours to the assay, and may fail if the sample contains other benign microorganisms which grow more rapidly than the pathogen. To overcome this problem, a new method for concentrating pathogens in food samples has been developed. Small glass beads coated with antibodies against the pathogen (similar to those naturally produced in the body to attack bacteria) are packed into a tube or column, and the sample is allowed to flow over the beads. The pathogens stick to the antibodies and are captured, while the rest of the sample passes out of the column. By concentrating the pathogens into a small volume (less than a drop of water), this process serves the same purpose as enrichment, but is much faster. The effect of bead size and sample flow rate was studied to determine the optimum conditions for capture. Future research will link this concentration process to a number of existing detection methods to produce rapid, sensitive pathogen assays. Technical Abstract: Affinity phases were prepared by coupling biotin or anti-E. coli O157 antibody to glass beads of defined diameter ranging from 35 micrometers to 138 micrometers. Micro-columns containing 20 microliters of the beads were used to assess the effect of bead diameter on the capture of latex particles and bacteria onto the bead surface from liquid samples pumped through the columns at flow rates of 100 to 1500 microliters/min. Avidin-coated fluorescent latex particles (1 micrometer diameter) were used as surrogate bacteria with biotin-glass bead columns to provide rapid, accurate quantitation and visualization of capture. Capture efficiency was inversely proportional to bead size and flow rate; capture was spatially non-uniform, with most captured particles found at the head of the column; and column capacity was greater than 100 particles/bead. Capture efficiency ranged from 95% (1500 microliters/min) to 99% (100 microliters/min) for 35 micrometers beads, and from 12% (1500 microliters/min) to 53% (100 microliters/min) for138 micrometer beads. E.coli O157:H7 captured on antibody-glass columns exhibited similar dependence on bead diameter and flow rate. This data will facilitate the optimization of micro-immunoaffinity techniques which must balance the need for high throughput (high flow rate), resistance to clogging by sample particulates (large beads), and high efficiency (small beads, low flow rates). |
