Figure 2

Binding of RG-1 antibody to disulphide hairpin of HPV16 L2. An HPV16 L2 13-31 cyclized peptide, purified to >99% homogeneity by HPLC, was completely reduced by the addition of 50 mM DTT and heating to 60°C. The redox state of the cyclized and reduced peptide was confirmed by mass spectrometry. The main neutral mass in the reduced sample was 2208.13Da and was 2Da larger than the 2206.13Da mass of the cyclized peptide. These main masses correspond to the expected masses of biotinylated HPV16 L2 13-31 peptide without and with disulfide bonds, respectively. The same day that the mass spectrometry was performed, the peptides were used to coat a microtiter plate. The binding of dilutions of RG-1, or a rabbit polyclonal antibody raised against HPV16 L2 17-36 peptide fused to KLH [2], to the cyclized or reduced HPV16 L2 13-31 peptide was then compared by ELISA (B) using peroxidase-linked anti-mouse or anti-rabbit IgG secondary antibodies respectively.