ic absorbance never of each sample was calculated by subtracting LTR Neu absorbance from that of NIH3T3 cells. Antibody titer was estimated as the highest immune serum dilution generating a specific absorbance of 0. 5 at 492 nm. Sera titer is the mean value of individual serum ti ters. Individual serum samples from mice receiving rV neuT were randomly chosen. Individual V wt mouse serum was assayed at 1 250 dilution. Immunoglobulin subclasses were determined by ELISA using a Mouse Typer Isotyping Kit using individual serum of rV neuT vaccinated mice as previously described. For immunoprecipitation, cells were lysed in RIPA buffer containing 1% Triton 100, 0. 5% deo icolate, 0. 1% SDS, 20 mM Tris pH 7. 5, 150 mM sodium chloride, proteases and phosphatases inhibitors.
Protein concentration was de termined using the Bradford protein assay. Equal amounts of total proteins were immunoprecipitated using sera derived from different ani mals immunized with rV neuT or V wt, and Protein G sepharose overnight at 4 C. The immunoprecipitates were washed three times with RIPA buffer, boiled at 95 C and centrifuged to remove sepharose beads. The immunopre cipitates were separated by SDS PAGE and transferred into nitrocellulose membrane. After blocking with 5% non fat milk, the membrane was incubated with polyclonal anti neu antibody C18. After washing, the membranes were incu bated with goat anti rabbit secondary antibody HRP conjugated. The antigen antibody binding was visualized by chemiluminescence using SuperSignal West Pico Chemilu minescent Substrate kit.
Biologic activity of vaccinated mouse immune sera in vitro Antibody dependent cellular cytoto icity was in vestigated as previously described. BALB neuT SALTO tumor cells were used as targets, while spleen cells from normal BALB c mice were used as effectors at 50 1. Dilutions of sera pooled from AV-951 four mice vaccinated with 108 pfu rV neuT or V wt were assayed. Percentage of specific lysis was calculated as described. The results represent average percentage of cytoto icity of two independent e periments. Four ran domly chosen serum samples were pooled each time and used for two independent e periments. For cell proliferation of BALB neuT SALTO tumor cells, immunoglobulins from 108 rV neuT and V wt pooled sera were purified by protein G and dialyzed against PBS. Purity was determined by SDS PAGE and Coomassie blue staining.
SALTO cells were incubated in serum free DMEM containing 0. 2% BSA containing Igs. Igs were replenished every 24 h. All treatments were performed in triplicate. Survival of cells was assessed by the Sulforhodamine B cell proliferation assay. The percent change in relative cell number nevertheless was calculated as described by Yip et al. To analyze the ability of serum antibodies to induce down regulation of ErbB2 Neu receptor on SALTO tumor cells, indirect immunofluorescence under native conditions was performed. Briefly, cells were detached by incubation with 0. 02% EDTA in PBS and incubated with purifi