The TZM-bl assay was performed as described [41 (link)]. In brief, virus was added to cells in the presence of DEAE-dextran (Sigma), washed 1× on day 1 and luminescence was measured on day 2 using luciferase substrate Bright-Glo (Promega).
Human PBMC-based assay was performed as described [42 (link)]. Serially diluted SHIVIG was incubated with virus for 1 h at 37°C. The virus/SHIVIG mixture was then added to the cells. Supernatant aliquots were harvested every other day starting on day 3. To remove anti-Gag antibodies that could interfere with the p27 assay read-out, plates were washed 5 times on day 4. The levels of p27 in supernatants were assayed first in wells containing only cells plus virus. When p27 levels were in the linear phase of increase in these control wells, neutralization was assessed for test samples. To analyze the role of NK cells, the same PBMC assay was run with and without NK cells from the same donor. PBMC were depleted of NK cells using an anti-CD56 mAb linked to magnetic beads according to the manufacturer’s protocol (Stemcell Technologies).
Human PBMC-based assay was performed as described [42 (link)]. Serially diluted SHIVIG was incubated with virus for 1 h at 37°C. The virus/SHIVIG mixture was then added to the cells. Supernatant aliquots were harvested every other day starting on day 3. To remove anti-Gag antibodies that could interfere with the p27 assay read-out, plates were washed 5 times on day 4. The levels of p27 in supernatants were assayed first in wells containing only cells plus virus. When p27 levels were in the linear phase of increase in these control wells, neutralization was assessed for test samples. To analyze the role of NK cells, the same PBMC assay was run with and without NK cells from the same donor. PBMC were depleted of NK cells using an anti-CD56 mAb linked to magnetic beads according to the manufacturer’s protocol (Stemcell Technologies).
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