Peptide truncation and alanine scan.

Variant peptides with N- or C-terminal truncations and/or alanine substitutions were tested for their ability to block binding to the parent 20-mer peptides in an ELISA. In case the peptide in question contained an alanine, the alanine was replaced by a serine instead. Ninety-six-well plates were coated with 100 μl NeutrAvidin per well at a concentration of 0.5 μg/ml. The plates were incubated overnight at 4°C and washed 4 times with PBS plus 0.05% Tween 20. One hundred microliters of blocking buffer (PBS plus 10% FBS) was added to the plates, and the plates were incubated for 90 min at 4°C. Subsequently, the blocking buffer was discarded, 100 μl of biotinylated 20-mer peptide at 200 ng/ml was added to the plate, and the plates were incubated for 90 min at 4°C. Selected antibodies were simultaneously incubated with variant peptides. We used 30 μl/well of MAb at 600 ng/ml and incubated it with 30 μl/well of alanine-modified peptides at 100 μg/ml for 90 min at 4°C. After the plates were washed, 50 μl of the antibody-alanine peptide mix was added to the plate-bound peptides and the plates were incubated for 20 min at 4°C. The plates were washed, 100 μl/well of secondary goat anti-mouse IgGγ conjugated to HRP (diluted 1:1,000 in blocking buffer) was added, and the plates were incubated for 90 min at 4°C. A final wash step was performed, the plates were developed using o-phenylenediamine, and the OD₄₉₀ was read on a SpectraMax 250 spectrophotometer (Molecular Devices).