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Table 1 Intra-assay variability of IFNγ, IL2 and IFNγ + IL2 responses in the IFNγ/IL2 FluoroSpot assay

From: Validation of an IFNγ/IL2 FluoroSpot assay for clinical trial monitoring

Donor Antigen Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Replicate 6 Mean SD %CV
A
 S09 BZLF1 283 267 280 ND ND ND 277 8.50 3.07
 S11 BZLF1 432 440 493 ND ND ND 455 33.15 7.29
 S14 BZLF1 438 452 479 ND ND ND 456 20.84 4.57
 S16 BZLF1 458 374 384 ND ND ND 405 45.88 11.32
 S18 BZLF1 110 148 103 ND ND ND 120 24.21 20.12
 S01 EBNA3A 30 26 31 ND ND ND 29 2.65 9.12
 S10 EBNA3A 28 31 31 ND ND ND 30 1.73 5.77
 S12 EBNA3A 38 43 46 ND ND ND 42 4.04 9.55
 S20 EBNA3A 68 60 81 ND ND ND 70 10.60 15.21
 S21 EBNA3A 166 158 192 ND ND ND 172 17.78 10.34
 S12 PHA-L 35 35 45 44 Rejected 38 39 4.83 12.25
 S15 PHA-L 718 659 774 685 679 Rejected 703 45.01 6.40
 S21 PHA-L Rejected 715 836 740 786 771 770 46.20 6.00
 S25 PHA-L 293 326 391 250 276 Rejected 307 54.37 17.70
 S26 PHA-L 98 120 123 Rejected 98 123 112 13.20 11.75
 S27 PHA-L 253 280 284 264 226 Rejected 261 23.38 8.95
Mean          22.27 9.96
B
 S09 BZLF1 63 67 52 ND ND ND 61 7.77 12.80
 S11 BZLF1 28 27 30 ND ND ND 28 1.53 5.39
 S14 BZLF1 37 31 34 ND ND ND 34 3.00 8.82
 S16 BZLF1 39 39 43 ND ND ND 40 2.31 5.73
 S18 BZLF1 32 37 37 ND ND ND 35 2.89 8.17
 S01 EBNA3A 33 29 21 ND ND ND 28 6.11 22.08
 S10 EBNA3A 26 26 20 ND ND ND 24 3.46 14.43
 S12 EBNA3A 47 44 51 ND ND ND 47 3.51 7.42
 S20 EBNA3A 41 32 Rejected ND ND ND 37 6.36 17.44
 S21 EBNA3A 42 38 38 ND ND ND 39 2.31 5.87
 S12 PHA-L 142 148 143 142 112 Rejected 137 14.42 10.49
 S15 PHA-L 718 645 647 718 652 Rejected 676 38.43 5.68
 S21 PHA-L Rejected 685 688 693 725 700 698 16.02 2.29
 S25 PHA-L 413 410 408 382 Rejected 412 405 13.00 3.21
 S26 PHA-L 488 505 511 554 501 524 514 22.96 4.47
 S27 PHA-L 374 423 430 445 385 383 407 29.59 7.28
Mean          10.85 8.85
C
 S09 BZLF1 50 51 57 ND ND ND 53 3.79 7.19
 S11 BZLF1 19 18 21 ND ND ND 19 1.53 7.90
 S14 BZLF1 24 18 18 ND ND ND 20 3.46 17.32
 S16 BZLF1 26 23 26 ND ND ND 25 1.73 6.93
 S18 BZLF1 23 29 29 ND ND ND 27 3.46 12.83
 S01 EBNA3A 18 11 11 ND ND ND 13 4.04 30.31
 S10 EBNA3A 13 18 11 ND ND ND 14 3.61 25.75
 S12 EBNA3A 23 25 27 ND ND ND 25 2.00 8.00
 S20 EBNA3A 10 14 16 ND ND ND 13 3.06 22.91
 S21 EBNA3A 22 17 21 ND ND ND 20 2.65 13.23
 S12 PHA-L 0 0 0 0 0 0 0
 S15 PHA-L Rejected 317 372 345 363 Rejected 349 24.25 6.94
 S21 PHA-L Rejected 460 454 437 Rejected 489 460 21.65 4.71
 S25 PHA-L 113 118 124 Rejected 125 Rejected 120 5.60 4.66
 S26 PHA-L 66 79 89 68 70 93 78 11.43 14.75
 S27 PHA-L 175 181 200 189 142 Rejected 177 21.89 12.34
Mean          7.61 13.05
  1. Values represent the number of detected antigen-specific IFNγ (A), IL2 (B) and IFNγ + IL2 (C) SFC/2 × 105 PBMC (stimulated with 1 µg/ml BZLF1 or EBNA3A peptide pools) or mitogen-specific IFNγ (A), IL2 (B) and IFNγ + IL2 (C) SFC/5 × 104 PBMC (stimulated with 2 µg/ml PHA-L) in the IFNγ/IL2 FluoroSpot assay (data is not background subtracted); “rejected” wells were not accepted because they did not pass the quality control
  2. ND not done, SD standard deviation, CV coefficient of variation