Single Molecule Assay for Ultrasensitive Detection of Cathepsin B in Human Blood
ACS Omega | March 31, 2021
Thangavelu B and Boutté AM
ACS Omega 2021, 6, 14, 9609–9616
This study was performed using a Simoa Homebrew assay.
Cathepsin B (catB) is a lysosomal cysteine protease expressed in several cells and organs, where it plays a role in protein degradation and turnover. Extracellular, secreted catB has utility as a biomarker for a host of pathological or physiological states, including a myriad of cancers or neurological diseases and injuries. Analytical or diagnostic assessment may be limited by biological sample volume availability. Pathologically relevant changes in catB levels may occur at low-moderate concentrations that require accurate measurement to differentiate from basal levels. Furthermore, biological samples like plasma and serum, often occlude accurate catB measurements because of background and high variance, vastly limiting the ability to detect catB as a peripheral biomarker. Techniques for ultrasensitive protein detection that require low volumes of sample are necessary. To overcome these challenges, a digital enzyme-linked immunosorbent assay (ELISA) was developed for differential detection of catB within less than 5 μL of serum and plasma using the single molecule array (SiMoA) platform, which offers 1000-times more sensitivity and vastly reduced variance compared to colorimetric tests. In buffer, curve-fitting estimated the limit of detection (LoD) to be ∼1.56 and ∼8.47 pg/mL using two-step or three-step assay configurations, respectively. After correcting for endogenous levels, the estimated LoD was ∼4.7 pg/mL in the serum or plasma with the two-step assay. The lower limit of quantitation was ∼2.3 pg/mL in the buffer and ∼9.4 pg/mL in the serum or plasma, indicting the ability to measure small changes above endogenous levels within blood samples.
This study was performed using a Quanterix HD-1 Analyzer.