The reactivities of the synthetic HA peptides or purified HA0 protein (eENZYME LLC, Montgomery Village, MD) with the convalescent-phase sera from H1N1pdm patients and the serum samples from mice immunized with peptide conjugates were determined by ELISA. Briefly, each peptide (1 mg/well) or protein (0.1 mg/well) was used to coat 96-well microtiter plates (Corning Costar, Acton, MA) in 0.1 M carbonate buffer (pH 9.6) at 4uC overnight. After blocking with 1% bovine serum albumen (BSA), the plates were incubated with indicated diluted serum samples (human or mouse) at 37uC for 2 hr, then washed four times with PBS containing 0.1% Tween 20 (PBS-T). Bound IgG antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-human IgG or anti-mouse IgG (Sigma) at 37uC for 1 hr. After four washes with PBS-T, the reaction was visualized by addition of the substrate 3,39,5,59-tetramethylbenzidine (TMB, sigma). Color development was stopped by the addition of 50 ml/well of 2 M sulphuric acid after 15 min. The optical density at 450 nm (OD 450 nm ) was measured by an ELISA plate reader (Molecular Devices, Sunnyvale, California). To evaluate the reactivity of the P5-derived short peptides with the anti-P5 antibody, peptide-inhibition ELISA assays were performed by adding dilutions of the peptides to a constant amount of the antibody elicited by the P5-KLH conjugates (1:5000 dilution). Maximum binding to antigen-coated wells was observed in the absence of a peptide inhibitor. The antibody bound was expressed as a percentage of the maximum level of binding.