Resorufin β d galactopyranoside

Simoa assays were transferred to a plate-based ELISA for comparison. Capture antibody was diluted in ELISA Coating Buffer (BioLegend) at a concentration of 4 μg ml⁻¹, and a volume of 100 μl was coated per well on a Nunc MaxiSorp ELISA plate (BioLegend). Plates were incubated with capture antibody overnight at 4°C. Subsequently, the plate was washed three times with 200 μl PBST (0.5 ml Tween-20 in 1 l PBS). Sample was added to each well and incubated at room temperature for 3 h. The plate was again washed three times with 200 μl PBST. The corresponding detection antibody (100 μl) was added to the plate, which was left to incubate for 1 h. The detection antibody was then removed, and the plate was washed three times with 200 μl PBST. Streptavidin-labeled β-galactosidase (100 μl) from the Simoa Homebrew Assay Development kit (Quanterix) was then added, and the plate was incubated for 30 min. The plate was then washed five times with 200 μl PBST and incubated with 100 μl resorufin β-d-galactopyranoside, also from the Simoa Homebrew Assay Development kit (Quanterix), for 20 min in the dark. Plates were then imaged with a Tecan plate reader using Magellan version 7.2 software at excitation and emission wavelengths of 555 nm and 605 nm, respectively.