A sensitive and rapid chemiluminescence ELISA for filamentous bacteriophages.
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 approximately 50,000 particles/well. In this report, we present a sandwich ELISA that allows detection of approximately 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 degrees 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.
Shukla
Girja S
GS
Department of Surgery, Vermont Comprehensive Cancer Center, University of Vermont College of Medicine, Burlington, VT 05405, USA. Girja.Shukla@uvm.edu
Krag
David N
DN
D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001331:Automation; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D017912:Inovirus / Q000276:immunology / Q000302:isolation & purification*; D008163:Luminescent Measurements / Q000379:methods*; D019151:Peptide Library; D010455:Peptides / Q000276:immunology; D012680:Sensitivity and Specificity; D013696:Temperature
D019151:Peptide Library; D010455:Peptides