Figure 3. Conjugation to TMV confers immunizing activity to HA protein. Protein equivalent to 15 µg HA was injected subcutaneously in the right rear flank two weeks apart. (A) After a single dose (pV1), immunization, sera were analyzed for anti-HA antibodies at 10 d post injection by ELISA (solid bars, left Y axis). The data are presented as µg/ml anti HA IgG, compared with a known quantity of anti-HA-IgG (Sino Biological Inc.). Pre-existing anti-TMV IgG titers (patterned bars, right Y axis) are shown for the groups pre-immunized 2 times with 25 µg TMV (T+) before the start of TMV-HA immunizations. (B) After a second dose (pV2), IgG titers (solid bars, left Y axis) and HAI (patterned bars, right Y axis) were evaluated. Abbreviations: Glut (glutaraldehyde conjugated, light gray), EN (EDC/NHS conjugated, medium gray), SM (SMCC conjugated, dark gray), K-HA (KLH-HA conjugate), HA-HA (HA self-conjugate), T+ (pre immunized with TMV before vaccination), T-HA (TMV-HA conjugate).