Figure 6.

Hemocyanin N-deglycosylation affected the humoral response, carrier properties, and antitumor activity. A, effect of N-deglycosylation on the humoral immune response. The specific serum antibody titers of C57BL/6 mice that were immunized with 1 mg of native (N) or N-deglycosylated (D) hemocyanins were determined at day 45. Antibody titers against each hemocyanin form (native and N-deglycosylated) were estimated by ELISA, using an anti-mouse–IgG–FAL serum and pNPP. B, glycotope characterization of anti-native hemocyanin antibodies. The same protocol performed in A was developed with the sera from mice preincubated with a mix of glycans (mannose and galactose, 100 mm) and then with each hemocyanin. C, glycotope characterization of anti-N-deglycosylated hemocyanin antibodies. The assay was conducted similar to A with the corresponding antisera. D, effect of N-deglycosylation of hemocyanins on their carrier effect. The anti-DNFB titers of C57BL/6 mice immunized with 0.8 mg of native (N) or N-deglycosylated (D) hemocyanins coupled to DNFB were determined by ELISA using BSA-DNFB and revealed with an anti-mouse–IgG–FAL serum and pNPP. E, schedule of the antitumor experiment. Five to seven mice per group were primed with 400 μg of native and N-deglycosylated hemocyanins or PBS as a vehicle control. Two weeks later, they were challenged with B16F10 cells. In the following 12 days, 6 doses of 100 μg of hemocyanin were administered as immunotherapy. F, effect of N-deglycosylation of hemocyanins on the tumor growth. Tumor size was measured every 3 days until day 20 previous to the exponential growth of the tumor. Tumor volume was calculated using the ellipsoid formula. G, effect of N-deglycosylation of hemocyanins on survival of mice. Survival rates were followed up to 45 days. For all graphs, the results are representative of two independent experiments. Data are shown as the mean ± S.E. Analysis by t test. *, p < 0.05; **, p < 0.01.