The Deeper You Dig, The More You Find

“The deeper you dig, the more you find” is frequently heard in proteomics, particularly at Proteins Powering NexGen Healthcare—Revolutionizing Biomarker Research and Translational Science Symposium: Novel Tools and Latest Technologies, held October 29, 2015. The event was organized and sponsored by Quanterix (Lexington, Mass.) to introduce the Simoa HD-2 analyzer.

Simoa is a platform that automates fluorescence enzyme-linked immunoassays (ELISAs). Conventional ELISAs are run in tubes, which usually have reaction volumes in the microliter range. With Simoa, reaction volume is reduced to the femtoliter scale, providing 100‒1000× improvement in lower limit of detection (LLOD).

The seminar presented the HD-2 version, which has several significant upgrades over the HD-1, including greater reliability and precision in fluidics and software, and improved seals on the sample well plates. The capture wells are about 50% smaller, reducing reagent consumption by 80%. Software is now compliant with 21 CFR Part 11.

The ELISA starts with paramagnetic beads coated with a ligand that forms immunocomplexes. Beads are passed over an array of more than 200,000-fL wells. At very low concentration, only a few of the wells contain a particle. This is easily detected with a fluorescence imager since the reaction (i.e., dilution) volume is very small. Empty wells are dark. As the concentration increases, the number of wells with immunocomplexes increases. Twins and triplets eventually show up according to a Poisson distribution. A further increase in concentration results from fitting the signal from each well to the predicted average distribution using an algorithm developed by Quanterix.

The main advantage of the technology is that the improved detection limits facilitate detection and quantitation of proteins present in samples in the mid-femtogram or low-femtomolar range.

Kevin Hrusovsky, executive chairman and CEO of Quanterix, holds that enhanced detection sensitivity and quantitation of proteins are key to understanding the fine points of proteomics. Proteins are the key effectors of life, and many are present in very low concentrations. Thus, ultrasensitive protein assays are essential to discovering the biochemistry and biology of life and health.

With Simoa, multiplexing is possible, which enables assaying several proteins or extending the assay panel to include RNA and DNA in the same well. This is especially useful in epigenetics. Hrusovsky reported that half of the researchers who try an HD-1 analyzer purchase one. It is a significant investment—the instrument costs about $100,000 and the operating consumables (reagents, including antibodies and well discs) add $200,000/yr. Hrusovsky said that the Simoa HD-3 would be available in 2017, to be followed a year later with a point-of-care handheld model. He also promised that fluorescence detection will be augmented by a small mass spec detector.

Protein post-translational modification

Dr. Jennifer Van Eyk of Cedars-Sinai Medical Center (Los Angeles, Calif.) lectured on post-translational modification (PTM) of proteins, with a particular emphasis on cardiomyocytes and heart disease. To treat heart disease, the basic molecular pathway needs to be understood, and then drugs can be designed that may be as simple as a peptide fragment of Troponin I. Since single proteins can be isolated, it is meaningful to study the occupancy patterns of PTM such as phosphorylation. Dr. Van Eyk also touched on the need for a research program to study heart disease in women. Most cohorts for trials on heart disease are restricted to adult men. Female heart disease presents differently than it does in males, and may have significantly different diagnostic and therapeutic scenarios.

Blood‒brain barrier

The next lectures focused on the use of blood to measure trauma in the human brain. Dr. Robert Stern studies the relationship of sports injuries and Chronic Traumatic Encephalopathy (CTE) at Boston University (Mass.). Dr. Jessica Gill of the National Institutes of Health (Bethesda, Md.) discussed the trauma experienced by soldiers in the Iraq and Afghanistan wars. Stern and Gill see the difficulty in sampling brain biomarkers, which must pass the blood‒brain barrier (BBB). Hypothesizing that some markers do get through, but that the quantities are low and diluted by large volumes of blood and cerebrospinal fluid (CSF), they have interest in Simoa, with its 1000× improvement in LLOD.

Dr. Stern focused on professional athletes such as boxers, and football, soccer and hockey players, who often endure chronic severe head trauma. In one study, about a third of a cohort of 20 professional linemen from the National Football League showed little sign of trauma-induced damage, while the remainder showed measurable-to-severe loss of mental function. Retrospective analysis of the small cohort correlates with a specific gene. The function of the gene is not known, and, since the sample size is small, the correlation may be coincidental. This observation will be investigated further in a large clinical trial program called DETECT (Detecting Deterioration, Evaluation, Treatment, Escalation and Communicating in Teams). Stern expects that CTE diagnostics will involve a panel of proteins.

Initially, in Dr. Stern’s investigation of CTE, large deposits of tau protein tangles in the brains of afflicted players were revealed during autopsy. Tau proteins usually protect microtubules, including the myelin sheath of neurons, but the frayed morphology of the patients’ tau was completely different from the smooth, ordered structure of the controls. Dr. Stern proposed that the brain tau had been disrupted by some type of prion-like mechanism.

An audience member stated that tau is produced at low levels by other organs. However, with Simoa, he is now able to detect brain taus in plasma (see below).

Tau proteins
Tau proteins stabilize microtubules. They are abundant around neurons of the central nervous system, and are expressed at very low levels in CNS astrocytes and oligodendrocytes. Tau proteins are the product of alternative splicing from the MAPT (microtubule-associated protein tau) located at chromosome 17q21. In the human brain, six isoforms of tau proteins exist. Three isoforms have three biding domains, and the other three have four domains. All binding domains are located at the carboxy terminus and carry a positive charge, which aids binding to the negatively charged microtubules. Highly phosphorylated tau protein correlates well with taupathies, including Alzheimer’s disease.

Dr. Gill reported on her work characterizing traumatic brain injuries (TBIs) suffered by a cohort of the 400,000 American soldiers deployed to Iraq. Some suffer multiple TBIs. She cautioned that, while military TBIs are very visible, an additional 2 million Americans suffer from TBIs due to sports, falls and automobile accidents.

The patient load is large. With time, about 80% recover, and the rest have serious long-term problems. Since the impact is so great, there is a need for diagnostics and effective therapy.

Dr. Gill presented data on some biomarkers like brain tau at fixed time points after injury. While her report reflected a work in progress, she and her team now have the analytical ability to detect difficult to find biomarkers.

Biomarkers for asthma

Developing therapeutics for asthma is complicated for several reasons, explained Dr. Monica Gavala of the Amgen Inflammation Therapeutic area (South San Francisco, Calif.). Drugs that seem to be effective in a small cohort fail to show efficacy in Phase 3 trials. The working hypothesis is that the multivalent nature of asthma dilutes effective response of a few with a huge cohort of nonresponders. Thus there is a need for precision medicine involving companion drugs and diagnostics.

Gavala’s group has been using an HD-1 analyzer since late 2014. Assays for IL-6 and IL-17A have delivered the expected improvement for lower limit of detection and quantitation.

User evaluation

Dr. Jane Ruppel, lead scientist at Genentech (South San Francisco, Calif.), used Simoa technology for assay development. Starting with the IL-17FF kit, she investigated precision, sensitivity, specificity and reliability. She advised that nonspecific adsorption can be a problem. In addition, as the analyte concentration decreases, other issues, particularly interferences, become important. She wanted an orthogonal mass spec assay for validation with isotopically labeled analytes, which correlated well.

Clinical diagnostics

Simoa tests are currently marketed for research use only (RUO). For clinical diagnostics, BioMérieux (Marcy-I’Etoile, France) has been licensed to work with regulatory agencies such as the FDA to get marketing approval for diagnostics.

For more information on Simoa technology, visit www.quanterix.com

Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected].

 


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