Efficient and reproducible new semimicromethod for the detection and titration of HIV in human plasma

Abstract

A semimicromethod was established for isolating human immunodeficiency virus (HIV) in plasma using 48-well plates and a pool of peripheral blood mononuclear cells (PBMC) from several donors as targets for infection, which increases the efficiency of isolation by reducing the effect of variability due to diverse donor cell susceptibility to HIV infection. The addition of H9 cells to the PBMC cultures did not affect measurable titers. Nevertheless, it potentiated strongly virus replication in terms of p24 production in the supernatant of the wells with HIV isolates, thus facilitating interpretation of the results. The titration of a virus strain of a known titre and reverse transcriptase activity in parallel provided a constant parameter of efficiency and reproducibility within each experiment, permitting comparison with results from other laboratories. The reproducibility of the method was highly significant (r = 0.97, P < 0.001); 68% of the 22 plasma samples from HIV-infected individuals tested by this method were positive. The presence of plasma HIV titer correlated well (P < 0.02) with the low count of CD4+ cells of less than 300/mm3, but not with the presence of the p24 antigen in the serum.

Human Immunodeficiency Virus Type 1 Infection Modulates the Interleukin (IL)–1β and IL‐6 Responses of Human Macrophages to CD40 Ligand Stimulation

“…These strains were isolated from plasma in PBMC cultures; the supernatants of these cultures were used as the source of the virus. Titration to determine infectivity of Ba-L and primary isolates was done, respectively, in a primary macrophage system or in PBMC as described elsewhere [22,23]. The titers of the virus stocks, expressed as TCID 50 , were determined as described elsewhere [24].…”

HIV-1 does not alterin vitroandin vivoIL-10 production by human monocytes and macrophages

“…Isolation of these strains from the plasma was performed in PBMC cultures, and the supernatants of these cultures were used as the source of the virus. Titration to determine infectivity of Ba-L and primary isolates was performed, respectively, in a primary macrophage system or in PBMC as previously described [33,34]. The titre of the virus stocks, expressed as 50% tissue culture infectious dose (TCID 50 ), was determined as previously described [35].…”

Increased CD4 and CCR5 Expression and Human Immunodeficiency Virus Type 1 Entry in CD40 Ligand–Stimulated Macrophages

“…The strains (FB1, FB2, and FB3) replicate in primary lymphocytes and macrophages but not in transformed cell lines. Titration to determine infectivity of HIV-1 Ba-L and of primary isolates, respectively, was performed in a primary macrophage system and in PBMC, as described elsewhere [25,26]. The titer of the virus stocks, expressed as TCID 50 , was determined, as described elsewhere [27].…”