Table 2.

Validation results of immunoassays for VAMP2 and SNAP25

Parameter	Acceptance criteria	VAMP2 ELISA				SNAP25 Simoa
Sensitivity – LLOQ Conc	Meanblank + 10*SDblank	127 pg/ml				2.91 pg/ml
Precision	CV < 15%	Conc	Intra-assay		Inter-assay	Conc	Intra-assay		Inter-assay
Low sample		235	6.3%		6.3%	4	8.3%		10.6%
Medium sample		299	2.6%		4.5%	6	14.3%		14.3%
High sample		748	3.5%		3.5%	10	7.9%		7.9%
		Mean	4.1%		4.8%	Mean	10.2%		10.9%
Dilutional linearity	80–120% linearity	Mean 3 samples				Mean 3 samples
		DF (x)		%L		DF (x)		%L
		1		-		1		-
		2		155		3		108
		4		107		6		112
		8		106		16		115
		16		107		39		99
		32		97		98		97
		64		143
		Mean		110%		Mean		106%
Parallelism	80–120% in range with the slope of the calibrator	Mean 3 samples				Mean 5 samples
		Mean		SD		Mean		SD
R2		0.94		0.0263		0.98		0.005
Slope		1.54		0.109		1.41		0.071
Range		103%		7%		89%		4%

Partial validation of the immunoassays: novel VAMP2 ELISA and SNAP25 Simoa. All concentration values are in pg/mL. The table shows the LLOQ of the immunoassays; precision for 3 different CSF samples with low, medium, and high analyte concentrations as the mean %CV of samples intra-assay and inter-assay; the mean dilutional linearity for 3 CSF samples against their dilution factors; and the mean %parallelism with the calibrator for 5 CSF samples (SD, standard deviation; R², linear regression coefficient). VAMP2 ELISA: The mean intra- and inter-assay precision was 4.1% and 4.8% respectively, while the mean dilutional linearity was 110%. The mean parallelism on CSF samples was 103%. SNAP25 Simoa: The mean intra- and inter-assay precision was 10.2% and 10.9% respectively, while the mean dilutional linearity was 106%. The mean parallelism on CSF samples was 89%