A Sensitive and Rapid Chemiluminescence ELISA for Filamentous Bacteriophages

Abstract

Filamentous bacteriophage (Ff) displayed random peptide and antibody libraries are widely used to identify specific, high affinity, binding ligands. A critical element in the identification of target‐specific phages is to determine phage titers, not only at every round of selection, but also for normalizing phage titers of a set of individual clones for their comparative binding analysis. The conventional ELISA‐based Ff titration methods require a minimum of 4–5 hr assay time and their lowest reported detection limit is ∼50,000 particles/well.

In this report, we present a sandwich ELISA that allows detection of ∼1000 Ff particles/well in less than 2.5 hr. The values of correlation of coefficient (r²) for the curves at low phage concentrations (up to 10⁶ TU/well) were about 0.999 in our ELISA. Experiments conducted at different temperatures suggest using 40°C incubations when titering low phage concentration samples. Experiments were also conducted with conventional ELISA for comparison. Our ELISA method derives an advantage from using a chemiluminescence substrate that gives much larger signals and wide linear range of measurement, thus allowing discrimination between background and low Ff phage concentrations. In conclusion, the Ff titration method presented here is highly sensitive, rapid, and amenable to high throughput analysis.

Keywords:

• Filamentous phage
• Titration
• ELISA
• Phage‐display selection

Acknowledgments

The work was supported by The National Cancer Institute R01 CA80790, NIH CA22435 and, in part, by the S. D. Ireland Professorship of Oncology for Research.