4613347721

Neutralizmg antibody titration

Serum samples from mice bearing B16 tumors treated with VSV were collected at the indicated time points and tested for neutralizing activity using a plaąue reduction assay as described previously (Charan and Zinkemagel, 1986). Briefly, serial 2-fold dilutions of 1 40 prediluted serum samples were mixed with equal volumes of VSV (using the same mutant injected for the oncolytic treatment) containing 5 x 10³ PFU, and incubated for 90 min at 37°C. A total of 100 pi of this solution was transferred onto Vero celi monolayers in 96-well plates and incubated for 1 h at 37°C. Monolayers were overlaid with medium containing 1% methylcellulose, and incubated overnight at 37°C. Then the overlay was removed and the monolayer fixed and stained with 0.5% crystal violet dissolved in 5% formaldehyde, 50% ethanol, and 4.25% NaCl. The serum dilution reducing the number of plaąues by 50% was taken as titer. To determine IgG titers, undiluted serum was treated with an equal volume of 280 mM 2-ME for 1 h at room temperaturę before samples were processed, as described above. For cross-neutralization analysis, serum samples were mixed with VSV WT and processed as above.