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- W205653390 abstract "Abstract Human Interferon-gamma (IFN-y) ELISpot assays are designed for the detection of IFN-y secreting cells at the single cell level. We have developed and validated an ELISpot assay with enhanced sensitivity (SC) and compared its performance relative to a common commercial kit (CK). Three manufactured lots of ELISpot kits (SC1-3) and one CK were analyzed in cellular titration experiments using PHA stimulation of cryopreserved PBMC from four subjects. The minimum quantity of cells required to elicit a single spot-forming cell (SFC) per well was 25,000 for CK compared to 7,000 for SC. In many cases, > 50,000 cells were required to measure a response using the CK. Slopes of log/log plots were 2.3 for CK versus 1.7 for SC, with no overlap at the 95% CI level. At the upper detection limit (200,000 cells/well), the CK averaged 108±46 SFC/well, while SC 1-3 measured 264±79, 377±120 and 231 ±95 SFC/well, respectively, demonstrating a broader linear range. Differences in sensitivity and range are possibly due to spot size, SC produced spots 9 x 10-3 mm2 (95% CI 7-11) while CK averaged 19 x 10-3 mm2 (95% CI 17-21), P < 0.001. The same PBMC were also analyzed using CEF (CMV, EBV and Influenza A – specific peptide pools) and CMV peptide stimulants and media controls. For CEF, the mean values for SC 1-3 were 100.0, 90.0, and 86.6 SFC/well, vs. 77.7 for CK. The CMV responses were 104.3, 80.2 and 74.4 SFC/well for SC 1-3 vs. 77.1 for CK. PBMC added with no stimulus, gave backgrounds of <2 SFC/well in all cases. The average within run coefficients of variation (CV) were similar- 10.2, 14.1 and 18.9 for SC 1-3 and 13.8 for CK when stimulated using CEF and 20.8 to 26.4 with CMV. No significant differences were observed for means or variability between CK and SC 1-3. In summary, SeraCare has developed and validated an IFN-y ELISpot kit having an enhanced dynamic range and sensitivity." @default.
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- W205653390 date "2007-04-01" @default.
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- W205653390 title "Performance Evaluation of an Enhanced-Sensitivity Interferon-gamma ELISpot Assay Kit (132.5)" @default.
- W205653390 doi "https://doi.org/10.4049/jimmunol.178.supp.132.5" @default.
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