Capture of Escherichia coli O157:H7 Using Immunomagnetic Beads of Different Size and Antibody Conjugating Chemistry

Abstract

Immunomagnetic beads (IMB) were synthesized using anti-Escherichia coli O157 antibodies and magnetic beads of two different sizes (1 μm and 2.6 to 2.8 μm) that contained a streptavidin coating, activated carboxyl groups or tosylated surfaces. The synthesized IMB, together with a commercially available IMB, were used to capture different strains of E. coli O157:H7 and E. coli O157:NM. The E. coli capture was measured by the time resolved fluorescence (TRF) intensity using a sandwich assay which we have previously demonstrated of having a sensitivity of 1 CFU/g after 4.5 hour enrichment [1]. The analyses of measured TRF intensity and determined antibody surface concentration indicated that larger beads provided higher response signals than smaller beads and were more effective in capturing the target of interest in pure culture and ground beef. In addition, while each type of IMB showed different favorable capture of E. coli O157:H7, streptavidin-coated IMB elicited the highest response, on average. Streptavidin-coated IMB also provided an economic benefit, costing less than $0.50 per assay. The results could be used to guide the proper choice of IMB for applications in developing detection processes for E. coli O157:H7.

Keywords: Escherichia coli; antibody linkage; immunomagnetic beads; time-resolved fluorescence.

Figure 1.

Response obtained using 2.6–2.8 μm IMB in the capture of 1×10⁷ cells/mL of E. coli O157:H7 and E. coli O157:NM from suspension. A) shows the data obtained in counts per second (CPS) while B) shows the results normalized to the number of beads used per assay. The average percent error among replicates was ±7.8%.

Figure 2.

Response obtained using 1 μm IMB in the capture of 1×10⁷ cells/mL of E. coli O157:H7 and E. coli O157:NM from suspension. A) shows the data obtained in counts per second (CPS) while B) shows the results normalized to the number of beads used per assay. The average percent error among replicates was ±8.7%.

Figure 3.

Comparison of IMB using 1×10⁶ beads/20 μL assay volume. Each IMB was diluted in PBS to contain 1×10⁶ beads/20 μL and tested for capture with 1×10⁷ cells/mL E. coli O157:H7 strain B1409. The larger sized beads provided greater capture of bacteria than those of a smaller size. The average percent error among three independent experiments run in triplicate was ±23%.

Figure 4.

Capture of E. coli O157:H7 strain B1409 from ground beef. Strain B1409 was inoculated into ground beef at a concentration of 1 cfu/g, enriched for 24 h in mEC broth, and assayed with each IMB. A) shows the results obtained in counts per second, with all IMB producing a similar response (P = 0.64). However, when the data is normalized to the number of beads used per assay (B), the results were quite different with IMB-C, IMB-S, and IMB-T giving the greater signal. The data obtained is from three independent experiments run in duplicate. The average percent error among these replicates was ±22%.