The TZM-bl neutralization assay was used to quantify NAb breadth as previously described [45] (link). Briefly, 500 infectious pseudovirus particles, as determined by the infectious titer described above, were incubated in duplicates with 2-fold serial dilutions of plasma for 1 hour, beginning with an initial concentration of 1∶100, before 10,000 TZM-bl reporter cells per well were added. Each plasma-virus combination was tested in duplicate and the assay was repeated twice. Infection levels were determined by B-galactosidase activity after 48 hours using a chemiluminescent readout. The IC50, or reciprocal plasma dilution at which 50% of the virus is neutralized, for each plasma-virus pair was calculated using linear interpolation from the neutralization curve. In this assay, a plasma sample was considered to be below the detectable limit of neutralization for a given virus if the lowest dilution (1∶100) did not show >50% neutralization. With this criterion, plasma samples that showed neutralization below the limit of detection were designated an IC50 value of 50, the midpoint between our starting dilution (1∶100) and 0. Each round of assays included HIV-negative plasma and a HIV-positive plasma pool from 30 HIV-1 infected individuals in Kenya between 1998–2000 [45] (link) serving as negative and positive internal controls, respectively. If a run showed neutralization of the negative control virus (SIVmne CL8) greater than the limit of detection, we considered that a failed run and repeated the assay.
Free full text: Click here
