Toward the quantification of α-synuclein aggregates with digital seed amplification assays.

The quantification and characterization of aggregated α-synuclein in clinical samples offer immense potential toward diagnosing, treating, and better understanding neurodegenerative synucleinopathies. Here, we developed digital seed amplification assays to detect single α-synuclein aggregates by partitioning the reaction into microcompartments. Using pre-formed α-synuclein fibrils as reaction seeds, we measured aggregate concentrations as low as 4 pg/mL. To improve our sensitivity, we captured aggregates on antibody-coated magnetic beads before running the amplification reaction. By first characterizing the pre-formed fibrils with transmission electron microscopy and size exclusion chromatography, we determined the specific aggregates targeted by each assay platform. Using brain tissue and cerebrospinal fluid samples collected from patients with Parkinson's Disease and multiple system atrophy, we demonstrated that the assay can detect endogenous pathological α-synuclein aggregates. Furthermore, as another application for these assays, we studied the inhibition of α-synuclein aggregation in the presence of small-molecule inhibitors and used a custom image analysis pipeline to quantify changes in aggregate growth and filament morphology.

Ultrasensitive Protein Detection Technologies for Extracellular Vesicle Measurements.

Extracellular vesicles (EVs) are nanoscopic, heterogenous, lipid-rich particles that carry a multitude of cargo biomolecules including proteins, nucleic acids, and metabolites. Although historically EVs were regarded as cellular debris with no intrinsic value, growing understanding of EV biogenesis has led to the realization that EVs facilitate intercellular communication and are sources of liquid biomarkers. EVs can be isolated and analyzed from a wide variety of accessible biofluids for biomarker discovery and diagnostic applications. There is a diversity of EVs from different biological compartments (e.g., cells and tissues), and some of these EVs are present at extremely low concentrations. Consequently, a challenge in the field is to find appropriate markers that enable selective isolation of these rare EVs. Many conventional protein detection technologies have limited sensitivity to detect low abundance biomarkers in EVs, limiting their use in EV research. Advances in ultrasensitive detection technologies are needed to harness the potential of EVs for clinical application. This Perspective highlights current EV research focusing on ultrasensitive detection technologies, their limitations, and areas of potential growth in the future.

High Frequency of Prior SARS-CoV-2 Infection by Sensitive Nucleocapsid Assays.

Prior infection with SARS-CoV-2 is typically measured by nucleocapsid serology assays. In this study, we show that the Simoa serology assays and T cell intracellular cytokine staining assays are more sensitive than the clinical Elecsys assay for detection of nucleocapsid-specific immune responses. These data suggest that the prevalence of prior SARS-CoV-2 infection in the population may be higher than currently appreciated.

Circulating Spike Protein Detected in Post-COVID-19 mRNA Vaccine Myocarditis.

BACKGROUND: Cases of adolescents and young adults developing myocarditis after vaccination with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-targeted mRNA vaccines have been reported globally, but the underlying immunoprofiles of these individuals have not been described in detail. METHODS: From January 2021 through February 2022, we prospectively collected blood from 16 patients who were hospitalized at Massachusetts General for Children or Boston Children's Hospital for myocarditis, presenting with chest pain with elevated cardiac troponin T after SARS-CoV-2 vaccination. We performed extensive antibody profiling, including tests for SARS-CoV-2-specific humoral responses and assessment for autoantibodies or antibodies against the human-relevant virome, SARS-CoV-2-specific T-cell analysis, and cytokine and SARS-CoV-2 antigen profiling. Results were compared with those from 45 healthy, asymptomatic, age-matched vaccinated control subjects. RESULTS: Extensive antibody profiling and T-cell responses in the individuals who developed postvaccine myocarditis were essentially indistinguishable from those of vaccinated control subjects, despite a modest increase in cytokine production. A notable finding was that markedly elevated levels of full-length spike protein (33.9±22.4 pg/mL), unbound by antibodies, were detected in the plasma of individuals with postvaccine myocarditis, whereas no free spike was detected in asymptomatic vaccinated control subjects (unpaired t test; P<0.0001). CONCLUSIONS: Immunoprofiling of vaccinated adolescents and young adults revealed that the mRNA vaccine-induced immune responses did not differ between individuals who developed myocarditis and individuals who did not. However, free spike antigen was detected in the blood of adolescents and young adults who developed post-mRNA vaccine myocarditis, advancing insight into its potential underlying cause.

L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma.

L1CAM is a transmembrane protein expressed on neurons that was presumed to be found on neuron-derived extracellular vesicles (NDEVs) in human biofluids. We developed a panel of single-molecule array assays to evaluate the use of L1CAM for NDEV isolation. We demonstrate that L1CAM is not associated with extracellular vesicles in human plasma or cerebrospinal fluid and therefore recommend against its use as a marker in NDEV isolation protocols.

Detectable plasma severe acute respiratory syndrome coronavirus 2 spike antigen is associated with poor antibody response following third messenger RNA vaccination in kidney transplant recipients.

BACKGROUND: Kidney transplant recipients (KTRs) generate lower antibody responses to messenger RNA (mRNA)-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination, yet precise mechanisms for this poor response remain uncertain. One potential contributor is suboptimal spike antigen (sAg) translation and expression owing to transplant immunosuppression, which might lead to insufficient exposure to develop humoral and/or cellular immune responses. METHODS: Within a single-arm clinical trial, 65 KTRs underwent ultrasensitive plasma sAg testing before, and 3 and 14 days after, the third mRNA vaccine doses. Anti-SARS-CoV-2 spike antibodies (anti-receptor binding domain [anti-RBD]) were serially measured at 14 and 30 days post-vaccination. Associations between sAg detection and clinical factors were assessed. Day 30 anti-RBD titer was compared among those with versus without sAg expression using Wilcoxon rank sum testing. RESULTS: Overall, 16 (25%) KTRs were sAg positive (sAg+) after vaccination, peaking at day 3. Clinical and laboratory factors were broadly similar in sAg(+) versus sAg(-) KTRs. sAg(+) status was significantly negatively associated with day 30 anti-RBD response, with median (interquartile range) 10.8 (<0.4-338.3) U/mL if sAg(+) versus 709 (10.5-2309.5) U/mL if sAg(-) (i.e., 66-fold lower; p = .01). CONCLUSION: Inadequate plasma sAg does not likely drive poor antibody responses in KTRs, rather sAg detection implies insufficient immune response to rapidly clear vaccine antigen from blood. Other downstream mechanisms such as sAg trafficking and presentation should be explored.

New Views of Old Proteins: Clarifying the Enigmatic Proteome.

All human diseases involve proteins, yet our current tools to characterize and quantify them are limited. To better elucidate proteins across space, time, and molecular composition, we provide a >10 years of projection for technologies to meet the challenges that protein biology presents. With a broad perspective, we discuss grand opportunities to transition the science of proteomics into a more propulsive enterprise. Extrapolating recent trends, we describe a next generation of approaches to define, quantify, and visualize the multiple dimensions of the proteome, thereby transforming our understanding and interactions with human disease in the coming decade.

Persistent Circulating Severe Acute Respiratory Syndrome Coronavirus 2 Spike Is Associated With Post-acute Coronavirus Disease 2019 Sequelae.

The diagnosis of postacute sequelae of coronavirus disease 2019 (PASC) poses an ongoing medical challenge. To identify biomarkers associated with PASC we analyzed plasma samples collected from PASC and coronavirus disease 2019 patients to quantify viral antigens and inflammatory markers. We detect severe acute respiratory syndrome coronavirus 2 spike predominantly in PASC patients up to 12 months after diagnosis.

Otto S. Wolfbeis (1947-2023).

Otto Wolfbeis, Professor emeritus of Analytical Chemistry at the University of Regensburg, passed away on June 1, 2023. Along with his seminal work on optical sensors and fluorescent (nano)materials, he will be remembered as an outstanding researcher who inspired many talents around the world.

Single-molecule studies reveal method for tuning the heterogeneous activity of alkaline phosphatase.

Single-molecule-enzymology (SME) methods have enabled the observation of heterogeneous catalytic activities within a single enzyme population. Heterogeneous activity is hypothesized to originate from conformational changes in the enzyme that result from changes in the local environment leading to catalytically active substates. Here, we use SME to investigate the mechanisms of heterogeneous activity exhibited by tissue nonspecific alkaline phosphatase (TNSALP), which reveals two subpopulations with different catalytic activities. We show the effect of pH and temperature on the distribution of TNSALP activity and confirm the presence of two subpopulations attributed to half- and fully active TNSALP substates. We provide mechanistic insight about protein structure using molecular dynamic simulations and show pH- and temperature-dependent conformational transitions that corroborate experimentally observed changes in TNSALP activity. These results show the utility of SME to understand heterogeneous enzyme activity and demonstrate a simple approach using pH and temperature to tune catalytic activity within an enzyme population.

Circulating Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccine Antigen Detected in the Plasma of mRNA-1273 Vaccine Recipients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins were measured in longitudinal plasma samples collected from 13 participants who received two doses of mRNA-1273 vaccine. Eleven of 13 participants showed detectable levels of SARS-CoV-2 protein as early as day 1 after first vaccine injection. Clearance of detectable SARS-CoV-2 protein correlated with production of immunoglobulin G (IgG) and immunoglobulin A (IgA).

Severe Acute Respiratory Syndrome Coronavirus 2 Messenger RNA Vaccines in Allogeneic Hematopoietic Stem Cell Transplant Recipients: Immunogenicity and Reactogenicity.

The severe acute respiratory syndrome coronavirus 2 messenger RNA vaccine-induced humoral response and reactogenicity profile are described in allogeneic hematopoietic stem cell transplant (HSCT) recipients. Findings showed that 75.0% (by Simoa assay) or 80.0% (by Roche assay) of the HSCT cohort had a positive antibody response on series completion, compared with 100% in the healthy cohort.

Single-Molecule Enzymology for Diagnostics: Profiling Alkaline Phosphatase Activity in Clinical Samples.

Enzymes can be used as biomarkers for a variety of diseases. However, profiling enzyme activity in clinical samples is challenging due to the heterogeneity in enzyme activity, and the low abundance of the target enzyme in biofluids. Single-molecule methods can overcome these challenges by providing information on the distribution of enzyme activities in a sample. Here, we describe the concept of using the single-molecule enzymology (SME) method to analyze enzymatic activity in clinical samples. We present recent work focused on measuring alkaline phosphatase isotypes in serum samples using SME. Future work will involve improving and simplifying this technology, and applying it to other enzymes for diagnostics.

High-Sensitivity Single Molecule Array Assays for Pathological Isoforms in Parkinson's Disease.

BACKGROUND: Clinical trials for neurodegenerative diseases are increasingly utilizing measurements of post-translational modifications (PTMs) and pathological isoforms as surrogate markers of target engagement and therapeutic efficacy. These isoforms, however, tend to exist at femtomolar concentrations, well below the detection limit of conventional immunoassays. Therefore, highly sensitive and well-validated assays for these isoforms are needed. METHODS: We developed a novel panel of single molecule array assays for pathological isoforms and PTMs implicated in the development and pathophysiology of Parkinson's disease. We validated this panel by measuring these analytes in the cerebrospinal fluid of a cross-sectional cohort of 100 patients with Parkinson's disease and 100 healthy controls. RESULTS: When comparing patients with Parkinson's disease to healthy controls, alpha synuclein, pSer129 alpha synuclein, DJ-1, and C-reactive protein were shown to be reduced in patients with Parkinson's disease while p396 tau and neurofilament light chain were shown to be increased. A random forest analysis produced an area under the curve of 0.70 for the panel. CONCLUSIONS: Measurement of post-translational modifications and pathological isoforms in patients with Parkinson's disease improved diagnostic accuracy above that of total protein measurements, demonstrating the potential utility of these assays for monitoring patients in clinical trials.

Ultrasensitive Measurement of Both SARS-CoV-2 RNA and Antibodies from Saliva.

Tests for COVID-19 generally measure SARS-CoV-2 viral RNA from nasal swabs or antibodies against the virus from blood. It has been shown, however, that both viral particles and antibodies against those particles are present in saliva, which is more accessible than both swabs and blood. We present methods for highly sensitive measurements of both viral RNA and antibodies from the same saliva sample. We developed an efficient saliva RNA extraction method and combined it with an ultrasensitive antibody test based on single molecule array (Simoa) technology. We apply our test to the saliva of patients who presented to the hospital with COVID-19 symptoms, some of whom tested positive with a conventional RT-qPCR nasopharyngeal swab test. We demonstrate that combining viral RNA detection by RT-qPCR with antibody detection by Simoa identifies more patients as infected than either method alone. Our results demonstrate the utility of combining viral RNA and antibody testing from saliva, a single easily accessible biofluid.

Highly Sensitive and Multiplexed Protein Measurements.

Proteins are involved in many biological processes. Misfolded, truncated, or mutated proteins as well as over- or underexpressed proteins have been implicated in many diseases. Therefore, detection and quantification of proteins is extremely important. Conventional techniques such as the enzyme-linked immunosorbent assay, Western Blot, and mass spectrometry have enabled discovery and study of proteins in biological samples. However, many important proteins are present at low concentrations, rendering them undetectable using conventional techniques. Furthermore, limited ability to simultaneously measure multiple proteins in a sample has constrained our ability to fully study the proteome. In this review, we comprehensively discuss approaches for protein detection. We first discuss the fundamentals of proteins and protein assays, including affinity reagents, surface functionalization, assay formats, signal detection, and multiplexing. We then discuss the challenges with these methods and review existing methods for highly sensitive and multiplexed protein detection. Finally, we review recent advances in protein detection from the literature and discuss challenges and future directions.

Bottom-up single-molecule strategy for understanding subunit function of tetrameric ß-galactosidase.

In this paper, we report an example of the engineered expression of tetrameric ß-galactosidase (ß-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric ß-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10-4 per base). In addition, we studied the kinetics of the mistranslated ß-gal at the single-molecule level.

A SARS-CoV-2 Neutralization Assay Using Single Molecule Arrays.

Coronavirus disease 2019 (COVID-19) manifests with high clinical variability and warrants sensitive and specific assays to analyze immune responses in infected and vaccinated individuals. Using Single Molecule Arrays (Simoa), we developed an assay to assess antibody neutralization with high sensitivity and multiplexing capabilities based on antibody-mediated blockage of the ACE2-spike interaction. The assay does not require live viruses or cells and can be performed in a biosafety level 2 laboratory within two hours. We used this assay to assess neutralization and antibody levels in patients who died of COVID-19 and patients hospitalized for a short period of time and show that neutralization and antibody levels increase over time. We also adapted the assay for SARS-CoV-2 variants and measured neutralization capacity in pre-pandemic healthy, COVID-19 infected, and vaccinated individuals. This assay is highly adaptable for clinical applications, such as vaccine development and epidemiological studies.

Ultrasensitive Detection of Enzymatic Activity Using Single Molecule Arrays.

Enzyme assays are important for many applications including clinical diagnostics, functional proteomics, and drug discovery. Current methods for enzymatic activity measurement often suffer from low analytical sensitivity. We developed an ultrasensitive method for the detection of enzymatic activity using Single Molecule Arrays (eSimoa). The eSimoa assay is accomplished by conjugating substrates to paramagnetic beads and measuring the conversion of substrates to products using single molecule analysis. We demonstrated the eSimoa method for the detection of protein kinases, telomerase, histone H3 methyltransferase SET7/9, and polypeptide N-acetylgalactosaminyltransferase with unprecedented sensitivity. In addition, we tested enzyme inhibition and performed theoretical calculations for the binding of inhibitor to its target enzyme and show the need for an ultrasensitive enzymatic assay to evaluate the potency of tight binding inhibitors. The eSimoa assay was successfully used to determine inhibition constants of both bosutinib and dasatinib. Due to the ultrasensitivity of this method, we also were able to measure the kinase activities at the single cell level. We show that the eSimoa assay is a simple, fast, and highly sensitive approach, which can be easily extended to detect a variety of other enzymes, providing a promising platform for enzyme-related fundamental research and inhibitor screening.

Ultrasensitive Detection of Attomolar Protein Concentrations by Dropcast Single Molecule Assays.

Measurements of very low levels of biomolecules, including proteins and nucleic acids, remain a critical challenge in many clinical diagnostic applications due to insufficient sensitivity. While digital measurement methods such as Single Molecule Arrays (Simoa), or digital ELISA, have made significant advances in sensitivity, there are still many potential disease biomarkers that exist in accessible biofluids at levels below the detection limits of these techniques. To overcome this barrier, we have developed a simple strategy for single molecule counting, dropcast single molecule assays (dSimoa), that enables more target molecules to be counted through increased sampling efficiency and with a simpler workflow. In this approach, beads are simply dropcast onto a microscope slide and dried into a monolayer film for digital signal readout. The dSimoa platform achieves attomolar limits of detection, with an up to 25-fold improvement in sensitivity over Simoa, the current state of the art for ultrasensitive protein detection. Furthermore, due to its simple readout process and improved cost-effectiveness compared to existing digital bioassays, dSimoa increases amenability to integration into point-of-care platforms. As an illustration of the potential utility of dSimoa, we demonstrate its ability to measure previously undetectable levels of Brachyury, a tissue biomarker for chordoma, in plasma samples. With its significantly enhanced sensitivity and simplicity, dSimoa can pave the way toward the discovery of new biomarkers for early disease diagnosis and improved health outcomes.