Total TDP-43 quantifications in human CSF

The Single Molecule Array (SIMOA®; Quanterix Corporation, USA) technology was used for the development of high-sensitivity custom immunoassays using two proprietary murine IgG2a mAbs binding TDP-43: ACI-5891 and ACI-5965. ACI-5891 was conjugated to paramagnetic beads (Quanterix Corporation) as a capture reagent, and ACI-5965 was biotinylated (Thermo Fisher) to be used as a detection antibody. Assays were performed using a three-step protocol with all incubation steps performed at 25°C on a microplate shaker set at 800 rpm (Quanterix Corporation). The incubation times were 30 min with the antibody-conjugated capture beads, 10 min with the biotinylated detection antibody, and 10 min with streptavidin-β-d-galactosidase (SBG; Quanterix Corporation). Plates were washed between each incubation using a magnetic microplate washer (Quanterix Corporation) and read with a SR-X SIMOA® instrument (Quanterix Corporation). Assay optimizations were accomplished by adjusting the concentrations of mAbs either conjugated to the paramagnetic beads or after biotinylation. The molar ratio of biotin and linker used for mAb–biotin conjugations and the concentrations of SBG were also adjusted. Paramagnetic beads conjugated to the capture antibody were diluted in bead diluent (Quanterix Corporation), and human CSF samples were diluted in the general detector and sample diluent (Quanterix Corporation). SBG was diluted in SBG diluent (Quanterix Corporation). The lower limit of quantification was determined as the mean blank for a readout of average enzyme per bead (AEB) + 2.5 times the standard deviation. The lower limit of detection was set based on the signal at the lowest concentration of the calibrator (0.023 AEB). For calibrator, full length recombinant TDP-43 with a polyglycine tag in N-terminal was used (described above). The raw values in AEB rather than the extrapolated concentrations for CSF were used as the levels of TDP-43 signal was between lower limit of detection and lower limit of quantification.