. 2021 Dec 4;13:198. doi: 10.1186/s13195-021-00939-9

Table 1.

Analytical characteristics of the six P-tau assays

		P-tau181		P-tau181		P-tau181		P-tau217		P-tau231		P-tau231
Assay characteristics
Assay	Provider	Eli Lilly		ADx NeuroSciences		Quanterix		Eli Lilly		ADx NeuroSciences		Gothenburg (NA)
	Status	Prototype		Prototype		Commercial		Prototype		Prototype		Prototype
	Catalogue number	N/A		N/A		103714		N/A		N/A		N/A
	Biofluid	EDTA plasma		EDTA plasma		EDTA plasma		EDTA plasma		EDTA plasma		EDTA Plasma /Serum
Platform	Simoa	Simoa HD-X		Simoa HD-X		Simoa HD-X		Simoa HD-X		Simoa HD-X		Simoa HD-x/HD-1
Antibodies	Name capture	AT270		ADx252		AT270		FAb2 of IBA493		ADx253		ADx253
	Epitope capture (AA) according to tau 441 numbering	Sequence 176-PPAPKT(p)P-182 phosphorylated specifically at threonine-181		Phospho-Thr 181 and no cross-reactivity with phospho-Thr175		Sequence 176-PPAPKT(p)P-182 phosphorylated specifically at threonine-181		Peptide phosphorylated at Thr217		Phosphorylated tau at T231		Phosphorylated tau at T231
	Name detector	LRL		ADx204		Tau12		4G10E2		ADx204		Tau12
	Epitope detector (AA)	111–130 according to the Tau441 sequence		N-terminal, that recognizes all forms of tau except those phosphorylated at Tyr 18		N-terminal epitope 6-QEFEVMEDHAGT-18		111–130 according to the Tau441 sequence		N-terminal, that recognizes all forms of tau except those phosphorylated at Tyr 18		N-terminal epitope 6-QEFEVMEDHAGT-18
Assay protocol	Steps	2-step assay		2-step assay		2-step assay		3-step assay		2-step assay		3-step assay
	Incubation times, min	60-5		60-10.5		35-5		30-5.15-5.15		60-5.15		40-7-7
	Sample/calibrator volume, μL	100		135		100		100		100		100
	Beads volume, μL	25		25		25		25		25		25
	Detector volume, μL	20		20		20		100		20		100
	SBG volume, μL	100		100		100		100		100		100
Assay reagents	Helper beads, % of beads	66%		50%		60%		50%		50%		0%
	SBG, pM	150 pM		50pM		150 pM		150 pM		50 pM		300
	Detector, μg/mL	1		0.6		Unknown		0.1		0.6		2
Calibrator	Type	Synthetic peptide		Synthetic peptide		Recombinant protein		Synthetic peptide		Synthetic peptide		Recombinant protein
	No. of calibrator points	9		8		7		8		8		8
	Range, pg/mL	0.226-52		0.625-50		0.177-86		0.04-180		0.3125-40		0-64
	Curve fit	1/y²-weighted 5PL		1/y²-weighted 5PL		1/y²-weighted 4PL		1/y²-weighted 4PL		1/y²-weighted 5PL		1/y²-weighted 4PL
Sample dilution	Fold-dilution	4		5		4		2		5		2
Sample dilution	Recommended method	Automated		Manual		Automated		Automated		Manual		Automated
Assay sensitivity and precision results
Sensitivity	Analytical LLOQ, pg/mL	1.55		2.36		0.24		0.15		0.56		3.95
Sensitivity	Functional LLOQ, pg/mL	6.2		11.8		0.96		0.3		2.8		7.9
Concentrations of QC and KC panels	QC1: high, pg/mL	15		26.8		3.8		2		7.4		26.7
	QC2: intermediate, pg/mL	6.9		13.3		1.3		0.6		4.7		17.1
	QC3: low, pg/mL	5.9		9.6		1.1		0.2		2.6		8.3
	KC1, pg/mL	4.05		9.75		3.22		0.64		1.41		NA
	KC2, pg/mL	16.69		17.87		70.35		1.99		5.72		NA
	KC3, pg/mL	142.01		N/A		N/A		61.6		N/A		NA
Precision QCs	Average Intra-assay %CV	6.6		14.5		7.7		13.5		16.8		3.7
Precision QCs	Average Inter-assay %CV	10		15.2		19.5		14.1		27.7		5.1
Precision KCs	Average Intra-assay %CV	5.6		16		6		9		19.9		NA
Precision KCs	Average Inter-assay %CV	9		23		29.5		10.5		30.5		NA
Clinical samples measurements	Number	80		80		80		80		80		80
	Range concentration, pg/mL	2.69–21.65		1.91–77.29		0.89–8.65		0.04–1.93		1–16.07		5.68–25.8
	Within calibrator range, %	100%		100%		100%		100%		100%		100%
	Range, CV%	0.00–23.23		0.33–69.08		0.1–15.91		0.07–64		0.05–51.34		0.01–14.49
	Average CV%	5.74		12.20		5.83		14.20		8.25		3.35
	n measured <LLOQ	21		14		1		39		1		7
	n measured > 20%CV	1		13		0		16		3		0
Other validation results
Parallelism	Average slope of samples	0.67		0.43		0.49		0.59		0.61		0.90
	Range of slopes of samples	0.55–0.75		0.33–0.61		0.39–0.61		0.53–0.68		0.48–0.72		0.85–0.97
	Average slope of calibrator	0.67		0.39		0.52		0.60		0.72		0.78
	Parallelism, %	99.6		110.3		94.6		98.7		84		116
Dilution linearity	Spiked concentration, pg/ml	150		150		150		11		150
		Df (x)	Mean %L	Df (x)	Mean %L	Df (x)	Mean %L	Df (x)	Mean %L	Df (x)	Mean %L	Df (x)	Df (x)
	Linear dilution factor with mean %Linearity	1	-	1	-	1	-	1.00	-	1	-	1	1
		5	173	5	54	5	200	2.80	118	5	147	5	5
		25	132	25	139	25	117	7.84	125	25	81	25	25
		125	120	125	117	125	118	21.95	115	125	112	125	125
		625	294	625	376	625	153	61.47	109	625	150	625	625
		3125	426	3125	541	3125	265	172.10	134	3125	196	3125	3125
Recovery		Spike	Mean %R	Spike	Mean %R	Spike	Mean %R	Spike	Mean %R	Spike	Mean %R	Spike	Mean %R
	Spiked concentration (pg/mL) With mean %Recovery	0.8	102	0.85	54	0.8	72	0.4	108	0.85	131	1	150
		4.0	95	4.27	67	4.0	82	1	107	4.27	139	6	124
		20.0	149	21.33	67	20.0	83	4	113	21.33	147	24	113

Phospho-specific antibody FAb2 of IBA493 and anti-tau antibodies LRL and 4G10E2 are property of Eli Lilly and Company. Phospho-specific antibody ADx252, ADx253, and anti-tau antibody ADx204 are property of ADx NeuroSciences. Phospho-specific AT270 is of ThermoFischer Scientific, and Tau-specific Tau12 is of Sigma Aldrich. Analytical LLOQ was calculated as the mean signal of 16 blanks plus 10 times the SD, with the P-tau concentration extrapolated from the calibration curve. This was multiplied by the sample dilution factor to obtain the functional LLOQ. QC samples are EDTA plasma pools and specific to each assay. KC samples were from the providers and specific to each assay, either synthetic peptide or recombinant protein spiked in buffer (both Eli Lilly assays and P-tau181 Quanterix assay, respectively) or remnant EDTA plasma sample (both ADx assays). KCs were not available for the P-tau231 Gothenburg assay. Average intra-and inter-assay variation was derived from measuring the QC and KC panels over four independent runs (except only two runs for the high QC sample with the P-tau181 ADx). With each assay, 80 clinical samples were measured, but due to technical reasons duplicate results were obtained, for 66 samples with P-tau181 Eli Lilly, for 74 with P-tau181 ADx, for 79 with P-tau217 Eli Lilly, for 79 with P-tau231 ADx and for 74 with P-tau231 Gothenburg. No results were obtained for 3 samples with P-tau181 Eli Lilly, for 1 sample with P-tau181 ADx and for 3 samples with P-tau231 Gothenburg. For parallelism, with each assay, four samples were measured after being four-times 2-fold serially diluted (P-tau181 Eli Lilly, P-tau181 ADx, and P-tau231 ADx: starting dilution 5-fold, reaching 40-fold; P-tau181 Quanterix: starting dilution 4-fold, reaching 32-fold; P-tau217 Eli Lilly and P-tau231 Gothenburg: starting dilution 2-fold, reaching 16-fold). For dilution linearity, three samples were spiked with high recombinant protein concentration, subsequently measured undiluted, and serially diluted until low P-tau concentrations below the LLOQs of the assays. With the P-tau181 ADx assay, two out of three of the undiluted samples were not measurable, likely due to matrix effect. With the P-tau231 Gothenburg, signals were not detected for the lowest two dilutions with the three samples

P-tau Phosphorylated tau, SBG Streptavidin β-galactosidase, PL Polynomial, LLOQ Lower limit of quantification, QC Quality control, KC Kit control, CV Coefficient of variation, %L % linearity, %R % recovery, NA Not applicable