Figure 3.

Controls for the replication-competent lentivirus (RCL) assay: inoculation and acceptance criteria. (a) The initial version of the RCL assay utilized three negative controls setup at the start of the amplification and three at the start of the indicator phase. All cultures are assayed for RCL at the end of the indicator phase. Two sets of positive controls were utilized, both inoculating cultures with R8.71 attenuated HIV-1 virus at the TCID₅₀. Three cultures are set up at the start of the amplification phase and five cultures inoculated at the start of the indicator phase. Five cultures were chosen for the indicator phase positive control since virus expansion will only be occurring for 7 days and the level of virus after this time is expected to be low. However, both the amplification phase positive controls and the indicator phase positive controls must have at least one culture positive for RCL for the assay to be valid. The assay is considered positive is the p24, psi-gag PCR, or both are positive. The established acceptance required all negative cultures to be negative for RCL. (b) The RCL assay was modified to remove the indicator phase negative and positive controls. The inoculum at the start of the amplification phase was increased to 5 IU per positive culture. An inhibitor control was added to the assay in which the test articles (at a concentration ≤1,000 ng/ml of p24) are spiked with 50 IU. At least one of the amplification phase positive controls and two of the inhibitor controls must be positive for the assay to be valid. IP, intraperitoneal.