Table 3.
Human IFN-γ ELISpot assay validation summary
| Assay parameter | Acceptance criteria | Validation results |
|---|---|---|
| LOD | the LOD is calculated using the 95th percentile of the mock-stimulation responses | LOD = 60 SFU/million PBMCs |
| CCP | the confirmatory cutpoint is calculated using the 95th percentile of the peptide/mock-stimulation response ratios | Confirmatory cutpoint = 2.96 response ratio |
| PHA minimum | the lower acceptance limit for a test sample under PHA stimulation is calculated empirically as the 1st percentile | PHA response ≥150 SFU/million PBMCs |
| Intra-triplicate precision | intra-triplicate CV ≤30% for responses ≥30 SFU/well, for each donor and peptide pool | Intra-triplicate CV average: |
| Low to medium response level: 10.8% | ||
| High response level: 4.2% | ||
| Intra-assay precision | intra-assay CV ≤30% for responses ≥30 SFU/well, for each donor and peptide pool | intra-assay CV average: |
| low to medium response level: 7.6% | ||
| high response level: 3.2% | ||
| Interassay precision | interassay CV ≤50% for responses ≥30 SFU/well, for each donor and peptide pool | interassay CV average: |
| low to medium response level: 35.9% | ||
| high response level: 27.4% | ||
| LLOQ | report the lowest peptide response that shows an intra-triplicate CV ≤30% in 2 independent sample preparations in at least 5 of 6 runs | 222 SFU/million PBMCd |
| ULOQ | report the 95th percentile of the highest peptide responses at which single spots can be detected and intra-triplicate CV ≤30% | 4,822 SFU/million PBMCs |
| Dilution linearity | report the range of cell densities through which a linear regression of responses shows an R2 ≥0.90 | 50,000–400,000 PBMCs/well |
| Specificity | no positive response to irrelevant control peptides or previously non-stimulating peptides in ≥5 of 6 donors | no cross-reactivity to HIV peptides or previously non-stimulating peptides was observed in 6 of 6 donors |
| Stimulant concentration robustness | the response for each robustness condition should be within |RE| ≤ 30% of the response observed under the standard conditions | 2 μg/mL; assays performed using less than the above-stated concentration of stimulating peptides were not robust |
| PBMC stimulation time robustness | the response for each robustness condition should be within |RE| ≤30% of the response observed under the standard conditions | 20–24 h; assays performed at 16 h incubation time were not robust |
| Substrate development time robustness | the response for each robustness condition should be within |RE| ≤30% of the response observed under the standard conditions | 3–4 min; assays performed at 2-min substrate development time were not robust |
| Plate drying time robustness | the response for each robustness condition should be within |RE| ≤30% of the response observed under the standard conditions | 20 h–80 days |
| Long-term PBMC sample stability | passing performance of a control donor sample was monitored over time during sample testing | up to 18 months, if stored in the vapor phase of LN2 |
CCP, confirmatory cutpoint; CV, coefficient of variation; LLOQ, lower limit of quantitation; LN2, liquid nitrogen; LOD, limit of detection; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin-L; SFU, spot-forming units; ULOQ, upper limit of quantitation.