An Ultrasensitive Assay for Measurement of Levels of Cathepsin B in the Blood

A novel digital enzyme-linked immunosorbent assay (ELISA) has been developed for the differential detection of cathepsin B from samples of serum or plasma.

Cathepsin B (catB) is a lysosomal cysteine protease expressed in various cells and organs, where it plays a role in protein degradation and turnover. Under pathological conditions, catB expression becomes upregulated in a variety of diseases including metastatic cancers, infections, traumatic brain injury, and neurological diseases. This upregulation is often associated with increased extracellular secretion via active or passive mechanisms. As such, the catB protein content is elevated above basal levels where it may have utility as a biofluid-based marker of diseases, injury, or trauma.


While biofluid samples such as plasma and serum contain catB, it is often difficult to obtain accurate measurements of the protein due to background interference and high variance, which limit the usefulness of catB as a peripheral biomarker. Thus, techniques for ultrasensitive protein detection that require low volumes of sample are necessary.

In this regard, investigators at Walter Reed Army Institute for Research (Silver Spring, MD, USA) developed a digital ELISA for differential detection of catB within less than five microliters 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.

Results revealed that in buffer solution, the limit of detection (LoD) was between 1.56 and 8.47 picograms per milliliter depending on whether a two-step or three-step assay was used. After correcting for endogenous levels, the estimated LoD was approximately 4.7 picograms per milliliter in serum or plasma with the two-step assay. The lower limit of quantitation was about 2.3 picograms per milliliter in buffer and about 9.4 picograms per milliliter in serum or plasma, indicting the ability to measure small changes above endogenous levels within blood samples.

"Although cathepsin can be abundant in some tissues, accurate measurement in blood has been a challenge, especially if changes are expected to be small or sample is limited," said first author Dr. Bharani Thangavelu, a researcher in the brain trauma neuroprotection branch at the Walter Reed Army Institute for Research. "Our strategy uses an ultrasensitive technique to improve cathepsin B detection from small volumes of blood with little to no noise or impact from interfering substances."

The ultrasensitive ELISA for cathepsin B was described in the March 31, 2021, online edition of the journal ACS Omega.

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Walter Reed Army Institute for Research