Detecting Single Proteins in Biological Matrices

Labcompare | February 16, 2017

Quanterix (Lexington, Mass.) was founded in 2007 with the goal of commercializing a diagnostic platform capable of measuring individual proteins at concentrations 1000 times lower than can be done using the best current immunoassays. The exclusive licensee of a broad intellectual property portfolio initially developed at Tufts University by Dr. David Walt, the company’s Simoa—or Single Molecule Array—platform enables the detection and quantification of single-molecule biomarkers in matrices such as plasma, serum, cerebrospinal fluid and cell extracts. This capability points to its potential for pharmaceutical companies, clinicians and researchers working in neurology, infectious disease research, cardiology, oncology and other areas. With Simoa, researchers can detect single protein molecules in biological matrices.

To begin an assay, antibody-capture agents are attached to the surface of 2.7-μm-diameter paramagnetic beads, each containing upwards of 250,000 attachment sites. Typically, 500,000 beads will be added to a 100-μL sample. At low target concentrations when the number of beads is greater than the number of target molecules in a sample, each bead will capture 0 or 1 target molecules. The fraction of beads that capture a target molecule in a given sample follows the Poisson Distribution. With so many beads in the solution, the bead-to-bead distance is so small that every molecule encounters a bead in less than a minute. At this time scale, diffusion of the target analyte molecules, even large proteins, occurs quickly, and in theory all the molecules should have multiple collisions with multiple beads, dramatically increasing binding.  Single molecules are trapped in femtoliter-sized wells on a Simoa Disc, which allows for a “digital” readout of each individual bead to determine if it is bound to the target analyte or not.

The beads are washed to remove nonspecifically bound proteins and are incubated with biotinylated detection antibody and then with β-galactosidase-labeled streptavidin. In this manner, each bead that has captured a single protein molecule is labeled with an enzyme. Beads that do not capture a molecule remain label-free. The beads are loaded into arrays of 216,000 femtoliter-sized wells, each of which hold no more than one bead. Beads are added in the presence of substrate, and wells are subsequently sealed with oil and imaged. If a target analyte has been captured (that is, an immune complex has formed), then the substrate will be converted to a fluorescent product by the captured enzyme label. The ratio of the number of wells containing an enzyme-labeled bead to the total number of wells containing a bead corresponds to the analyte concentration in the sample. By acquiring two fluorescent images of the array, it is possible to demonstrate an increase in signal and thus confirm the presence of a true immune complex: beads associated with a single enzyme molecule (an “on” well) can be distinguished from those not associated with an enzyme (an “off” well). The protein concentration in the test sample is determined by counting the number of wells containing both a bead and a fluorescent product relative to the total number of wells containing beads. Concentration is determined by digital ELISA.

 

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