The authors used a homogeneous calcium dye kit with a cell line transfected using a recombinant protein construct to screen a 50,000 compound library for G-protein coupled receptor (GPCR) agonists. Only 1 of the 365 primary hits activated Gq-coupled GPCRs, as shown using IP-ONE HTRF®. Furthermore, an agonist screen against the entire compound library and same heterologous cell line using AequoScreen™ technology generated no false positives and identified the same positive hit. Next, a multiplex assay composed of both Fluo-3 and Fura-2–loaded cells identified 1 false positive and the same true-positive hit out of the 365 primary hits. Finally, rescreening the 365 primary hits against the parental cell line loaded using the homogeneous calcium dye kit confirmed the specificity of the same true-positive hit only. In summary, the results suggest that AequoScreen™ technology, IP-ONE HTRF®, and multiplex assays are unique, orthogonal technologies to identify nonspecific hits.
The Ab-dependent cell-mediated cytotoxicity (ADCC) activity of anti-gp120 Abs in serum from four groups of HIV-1-positive individuals in the Multicenter AIDS Cohort Study was evaluated at several time points over a 10-yr period. HIV-1-positive individuals who progressed to AIDS within 3 yr of seroconversion (rapid progressors) were compared with seroconverters who did not progress to AIDS within 6 yr (nonrapid progressors) and individuals who were seropositive when they entered the study and did not progress to AIDS within 9-10 yr (nonprogressors). At the visit closest to AIDS, rapid progressors had significantly lower titers of Abs that mediate ADCC against HIV-1 gp120 than those of nonrapid progressors at corresponding visits or those of nonprogressors at any visit. Nonprogressors generally had high titers of ADCC Abs at all visits. Differences between ADCC titers of rapid progressors and nonrapid progressors or nonprogressors remained when longitudinal changes within individuals were compared. Among seroconverters who were nonrapid progressors, those with low or declining ADCC titers lost significantly more CD4+ cells during the study than those whose ADCC titers were stable or increasing, even though both groups had similar serum virion RNA levels. This demonstrates that high titers of Abs that mediate ADCC correlate with a successful host defense against AIDS.
While performing enzyme-linked immunosorbent assays (ELISAs) we have occasionally noted "geographical" effects on the ELISA plates, in which results from one region of the plate were considerably lower than those obtained on a different part of the plate. This effect would occur spontaneously, persist for a variable period of time and then disappear. Among the explanations we considered and excluded were variations in plate or reagent temperature, pipeting errors, unequal plate washing and residual reagent remaining in the wells. We became suspicious that static electricity might be affecting our results after observing the plastic wrap covering the plates was statically charged.
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