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

Körber, Nina; Behrends, Uta; Hapfelmeier, Alexander; Protzer, Ulrike; Bauer, Tanja

doi:10.1186/s12967-016-0932-7

Journal of Translational Medicine

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

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
ND not done, SD standard deviation, CV coefficient of variation

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ISSN: 1479-5876

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