Because of the compatibility of HTRF with high-throughput screening (HTS), “we decided to use the technology for the detection of cytokines,” explained Lorena Kallal, Ph.D., manager, biological reagents and assay development, at GlaxoSmithKline in Collegeville, PA. “In addition, you do not have to tag or engineer proteins to detect them if you have antibodies that specifically recognize your protein.”
Dr. Kallal, one of the speakers at the Avignon conference, noted that HTRF reagents tend to be less light sensitive and more stable than some other HTS-compatible detection systems.
“We also found that once reagents were added, we could get the same results if we read the plate a few hours after adding reagent, or overnight,” she continued. “For automated assays, it helps if the incubation times are flexible, and it also gives scientists flexibility in how they want to run the assays.”
Describing her team’s HTRF work with cytokines, she explained that since cytokines are secreted proteins, investigators have the option to test the supernatant only, to avoid interference from the cells in the well.
“We found that there was no difference in the IL-8 and IL-1b assays whether we tested the supernatant only, or whether we ran the assay on plates where the cells remained in the well,” explained Dr. Kallal. “And for one assay, we actually saw better statistics in 1,536-well plates than in 384 well plates.”
She added that although HTRF may not have the same sensitivity as some wash protocols, “if you have enough cytokine present, then there is no issue.”