Skip to main content

Table 1 Analytical characteristics of the six P-tau assays

From: Clinical and analytical comparison of six Simoa assays for plasma P-tau isoforms P-tau181, P-tau217, and P-tau231

  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
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
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
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
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
  1. 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
  2. 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