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

Table 2 Inter-assay variability of IFNγ, IL2 and IFNγ + IL2 responses in the IFNγ/IL2 FluoroSpot assay

Donor	Antigen	Plate 1	Plate 2	Plate 1 and plate 2
Donor	Antigen	Mean	Mean	Mean	SD	% CV
A
S09	BZLF1	274	233	254	28.99	11.44
S11	BZLF1	317	326	322	6.36	1.98
S12	EBNA3A	92	94	93	1.41	1.52
S13	EBNA3A	65	63	64	1.41	2.21
S15	PHA-L	645	680	663	24.75	3.74
S21	PHA-L	668	753	711	60.10	8.46
S25	PHA-L	234	287	261	37.48	14.39
S26	PHA-L	214	240	227	18.38	8.10
Mean					22.36	6.48
B
S09	BZLF1	56	47	52	6.36	12.36
S11	BZLF1	52	86	69	24.04	34.84
S12	EBNA3A	33	35	34	1.41	4.16
S13	EBNA3A	0	0	0	–	–
S15	PHA-L	575	657	616	57.98	9.41
S21	PHA-L	698	691	695	4.95	0.71
S25	PHA-L	411	403	407	5.66	1.39
S26	PHA-L	547	617	582	49.50	8.50
Mean					21.42	10.20
C
S09	BZLF1	52	49	51	2.12	4.20
S11	BZLF1	61	85	73	16.97	23.25
S12	EBNA3A	19	28	24	6.36	27.08
S13	EBNA3A	0	0	0	–	–
S15	PHA-L	329	349	339	14.14	4.17
S21	PHA-L	453	459	456	4.24	0.93
S25	PHA-L	86	105	96	13.44	14.07
S26	PHA-L	102	130	116	19.80	17.07
Mean					11.01	12.97

Values represent the mean number of 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/2 × 10⁵ PBMC/5 × 10⁴ PBMC (stimulated with 2 µg/ml PHA-L) detected in two different assay plates performed on the same day in parallel
SD standard deviation, CV coefficient of variation

ISSN: 1479-5876