Digital Enzyme-linked Immunosorbent Assay Research Articles

Serum and Salivary IgG and IgA Response After COVID-19 Messenger RNA Vaccination

There is still considerable controversy in the literature regarding the capacity of intramuscular messenger RNA (mRNA) vaccination to induce a mucosal immune response. To compare serum and salivary IgG and IgA levels among mRNA-vaccinated individuals with or without previous SARS-CoV-2 infection. In this cohort study, SARS-CoV-2-naive participants and those with previous infection were consecutively included in the CoviCompare P and CoviCompare M mRNA vaccination trials and followed up to day 180 after vaccination with either the BNT162b2 (Pfizer-BioNTech) vaccine or the mRNA-1273 (Moderna) vaccine at the beginning of the COVID-19 vaccination campaign (from February 19 to June 8, 2021) in France. Data were analyzed from October 25, 2022, to July 13, 2023. An ultrasensitive digital enzyme-linked immunosorbent assay was used for the comparison of SARS-CoV-2 spike-specific serum and salivary IgG and IgA levels. Spike-specific secretory IgA level was also quantified at selected times. A total of 427 individuals were included in 3 groups: participants with SARS-CoV-2 prior to vaccination who received 1 single dose of BNT162b2 (Pfizer-BioNTech) (n = 120) and SARS-CoV-2-naive individuals who received 2 doses of mRNA-1273 (Moderna) (n = 172) or 2 doses of BNT162b2 (Pfizer-BioNTech) (n = 135). The median age was 68 (IQR, 39-75) years, and 228 (53.4%) were men. SARS-CoV-2 spike-specific IgG saliva levels increased after 1 or 2 vaccine injections in individuals with previous infection and SARS-CoV-2-naive individuals. After vaccination, SARS-CoV-2-specific saliva IgA levels, normalized with respect to total IgA levels, were significantly higher in participants with previous infection, as compared with the most responsive mRNA-1273 (Moderna) recipients (median normalized levels, 155 × 10-5 vs 37 × 10-5 at day 29; 107 × 10-5 vs 54 × 10-5 at day 57; and 104 × 10-5 vs 70 × 10-5 at day 180 [P < .001]). In contrast, compared with day 1, spike-specific IgA levels in the BNT162b2-vaccinated SARS-CoV-2-naive group increased only at day 57 (36 × 10-5 vs 49 × 10-5 [P = .01]). Bona fide multimeric secretory IgA levels were significantly higher in individuals with previous infection compared with SARS-CoV-2-naive individuals after 2 antigenic stimulations (median optical density, 0.36 [IQR, 0.16-0.63] vs 0.16 [IQR, 0.10-0.22]; P < .001). The findings of this cohort study suggest that mRNA vaccination was associated with mucosal immunity in individuals without prior SARS-CoV-2 infection, but at much lower levels than in previously infected individuals. Further studies are needed to determine the association between specific saliva IgA levels and prevention of infection or transmission.

Development of a Companion Diagnostic for a Targeted Protein Degradation Therapy: Ultrasensitive and Minimally-Invasive Detection of SALL4 Oncoproteins for Therapeutic Monitoring

Abstract Introduction/Objective Spalt-Like Transcription Factor 4 (SALL4), a member of the SALL family, is a regulator of embryonic stem cell development that plays a crucial role in cell renewal and proliferation. SALL4 expression is normally repressed in most adult organs but is reactivated in a multitude of cancers and upon treatment with hypomethylating agents (HMA). Research in the past 20 years established SALL4 as a novel, druggable cancer target. SALL4 targeting therapies (inhibitors and degraders) are under pre-clinical development. There are currently no FDA/CLIA approved SALL4 diagnostic tests beyond tissue immunohistochemistry (IHC). Therefore, there is outstanding clinical need for the development and validation of a less invasive and sufficiently-sensitive SALL4 expression assay which can be used for selecting, and monitoring patients on SALL4-targeting therapy such as molecular glue degraders, as well as monitoring patients on HMA treatment. Methods/Case Report Using the Simoa (Single Molecule Array) platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 5 commercial SALL4 antibodies and 13 antibody pair combinations, using the best antibody pair to test the matrix effects of human plasma, buffy coats, and bone marrow aspirates. For quantitative assay development, we successfully expressed and purified full-length SALL4 which will be used to calibrate the standard curve and determine the assay’s limit of quantification. Results (if a Case Study enter NA) The best antibody pair can detect both SALL4A and SALL4B isoforms, with stronger signal detected with the SALL4A isoform. We successfully optimized detection of SALL4 in bone marrow and peripheral blood matrix, and tested the utility of the assay for quantifying the efficacy of SALL4 degrader drugs and with hypomethylating agent treatment, using cell culture system. We also optimized multiple pre-analytic steps, including our cell lysis protocol, isolation of buffy coats using density gradient separation, and sample collection method comparison. Conclusion We developed an ultrasensitive SALL4 expression assay will be useful for monitoring patients on HMA treatment and SALL4-targeting therapy such as molecular glue degraders. There is ongoing effort to use MGB Biobank peripheral blood samples to validate SALL4 Simoa for patients with myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML), and liver cancer.

Ultrasensitive Detection of SALL4 Oncoproteins in Peripheral Blood and Bone Marrow Aspirate: Companion Diagnostics and Therapeutic Monitoring

Spalt-Like Transcription Factor 4 (SALL4), a member of the SALL family, is a regulator of embryonic stem cell development that plays a crucial role in cell renewal and proliferation. SALL4 is a tumor driver, as shown by gain-of-function and loss-of-function studies; high SALL4 expression invariably correlates with worse prognosis. These studies established SALL4 as a novel, druggable target, such as in liver, lung, and blood cancers. In myelodysplastic syndrome (MDS), SALL4 can be upregulated by hypomethylating agents (HMAs), correlating with worse patient outcomes. Despite its well-established function on oncogenesis, SALL4 targeting therapies (inhibitors and degraders) are under pre-clinical development. There are currently no FDA/CLIA approved SALL4 diagnostic tests beyond tissue immunohistochemistry. Therefore, there is outstanding clinical need for the development and validation of a less invasive and sensitive SALL4 expression assay via peripheral blood, which can be used for monitoring patients on HMA treatment and SALL4-targeting therapy such as molecular glue degraders. We developed a protein based SALL4 assay, compatible with peripheral blood and bone marrow aspirates samples, using the SIMOA (Single Molecule Array) technology, a bead-based digital enzyme-linked immunosorbent assay (aka digital ELISA). Previously, circulating SALL4 oncoproteins have been detected in the peripheral blood of patients with liver cancer using a conventional ELISA kit, but it is only semi-quantitative with limited sensitivity. We cross-tested 5 commercial SALL4 antibodies and 13 antibody pair combinations. After antibody screening, we used the best antibody pair to test the matrix effects of human plasma, buffy coats, and bone marrow aspirates. For quantitative assay development, we successfully expressed and purified full-length SALL4 for the first time, which was used to calibrate the standard curve and determine the assay's limit of quantification. The top bead and detector antibody pair could detect both SALL4A and SALL4B isoforms, with stronger signal detected with the SALL4A isoform in two different cell lysis buffers. Assay sensitivity was then tested. The Simoa assay captured endogenous SALL4 expression with greater sensitivity compared to the Western blot. The Western blot's limit of detection for SALL4A protein was 0.08 µg/µL whereas the Simoa assay continued until 3 ng/µL. Pre-analytic steps were then optimized, including our cell lysis protocol, isolation of buffy coats using density gradient separation, and sample collection method comparison. We assessed the effect of sample age for downstream processing and signal recovery as well as the possible matrix effect and interference from the buffy coat and plasma components of the peripheral blood and bone marrow samples. Our data showed no evidence of interference from the peripheral blood components, and interference from bone marrow aspirate can be further optimized. Antibody validation was then conducted. The preliminary data showed that the SALL4 Simoa assay can be used as a proof of principle to monitor SALL4 upregulation on HMA treatment for blood cancers and for SALL4 targeting therapy with two cell lines (SNU398 and H661) for solid cancers. To validate assay accuracy and precision, a retrospective cohort analysis is being conducted from patient peripheral blood samples. We obtained MGB Biobank samples for MDS, acute myeloid leukemia (AML), and liver cancer. We will use these samples to test for digital SALL4 protein detection. Further plans include expanding these analyses to breast and lung cancer. Additionally, we will conduct prospective studies to explore the role of the SALL4 protein biomarker in risk stratification and treatment monitoring, especially in MDS/AML patients undergoing HMA treatments. In conclusion, we have developed an ultrasensitive SALL4 protein expression diagnostic assay for use in monitoring patient treatment response and for guiding targeted therapy by analyzing peripheral blood for both solid and liquid tumors. Future steps include validating the diagnostic assay for specificity and sensitivity in a retrospective cohort analysis and conducting prospective studies to explore the role of the SALL4 protein biomarker for risk stratification, monitoring on HMA treatment, and guiding targeted therapy, ideally filing for approval as a laboratory developed test (LDT) in the near future

Development of a quantitative thyroid-stimulating hormone assay system for a benchtop digital ELISA desktop analyzer.

Regular checkups for thyroid-stimulating hormone (TSH) levels are essential for the diagnosis of thyroid disease. The enzyme-linked immunosorbent assay (ELISA) technique is a standard method for detecting TSH in the serum or plasma of hospitalized patients. A recently developed next-generation ELISA, the digital immunoassay (d-IA), has facilitated detection of molecules with ultra-high-sensitivity. In this study, we developed a TSH assay system using the d-IA platform. By utilizing the ultrasensitivity of d-IA, we were able to use a sample volume of as little as 5µL for each assay (the dead volume was 5µL). The limits of blank, detection, and quantification (i.e., functional sensitivity), were 0.000346, 0.001953, and 0.002280μIU/mL, respectively, and the precision of the total coefficient of variation did not exceed 10%. The correlation between serum and plasma levels indicated good agreement. Thus, our system successfully measured TSH using d-IA with a small sample volume and equal functional sensitivity to the current third generation like ARCHITECT TSH assay, which has a functional sensitivity of 0.0038μIU/mL.

Improving the Accuracy, Robustness, and Dynamic Range of Digital Bead Assays.

We report methods that improve the quantification of digital bead assays (DBA)─such as the digital enzyme-linked immunosorbent assay (ELISA)─that have found widespread use for high sensitivity measurement of proteins in clinical research and diagnostics. In digital ELISA, proteins are captured on beads, labeled with enzymes, individual beads are interrogated for activity from one or more enzymes, and the average number of enzymes per bead (AEB) is determined based on Poisson statistics. The widespread use of digital ELISA has revealed limitations to the original approaches to quantification that can lead to inaccurate AEB. Here, we have addressed the inaccuracy in AEB due to deviations from Poisson distribution in a digital ELISA for Aβ-40 by changing the AEB calculation from a fixed threshold between digital counting and average normalized intensity to a smooth, continuous combination of digital counting and intensity. We addressed issues with determining the average product fluorescence intensity from single enzymes on beads by allowing outlier, high intensity arrays to be removed from average intensities, and by permitting the use of a wider range of arrays. These approaches improved the accuracy of a digital ELISA for tau protein that was affected by aggregated detection antibodies. We increased the dynamic range of a digital ELISA for IL-17A from AEB ∼25 to ∼130 by combining long and short exposure images at the product emission wavelength to create virtual images. The methods reported will significantly improve the accuracy and robustness of DBA based on imaging─such as single molecule arrays (Simoa)─and flow detection.

A novel method of digital ELISA using single molecule amplification enables sub-attomolar detection limits for blood-based proteomics and biomarker measurements

Abstract The application of measuring low-abundance protein biomarkers in blood (serum or plasma) can often present challenges due to limits of sensitivity or precision. Digital proteomics technologies such as the digital enzyme-linked immunosorbent assay (ELISA) have enabled 1000-fold increases in sensitivity over conventional immunoassay method. However, current digital ELISA technologies can lack sufficient sensitivity to quantitatively measure many low abundance proteins in blood samples. We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule amplification with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (5000 vs. 500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency). Using this approach, we characterize limit of detection (LOD) of of 10–100X more sensitive than the standard digital ELISA. A digital ELISA with improved sensitivity was used to measure cytokines in serum and plasma samples with values consistently above the assay lower limits of quantitation (LLoQ). The results were consistent with a kinetic model of binding that showed that combining capture antibodies with high on-rates with high antibodies per bead yields the greatest improvement in sensitivity. The novel approach to multiplex single molecule proteomics with attomolar sensitivity provides the potential to accelerate insights in proteomics biomarkers research, clinical applications and diagnostics.

Quantitative performance of digital ELISA for the highly sensitive quantification of viral proteins and influenza virus.

A single-molecule assay (SiMoA) using a digital enzyme-linked immunosorbent assay (ELISA) has been attracting attention as a promising method that can detect viruses with ultra-high sensitivity. However, the quantitative application of digital ELISA has not been adequately reported. Therefore, in this study, we first evaluated the linearity and sensitivity of digital ELISA using a Certified Reference Material of C-reactive protein (NMIJ CRM 6201-c) as a quality control material. Next, we originally screened those antibody pair that are suitable for detecting recombinant viral proteins of influenza A virus, nucleoprotein (NP), and hemagglutinin (HA), and established the measurement system. Under optimized conditions, the limit of detection (LOD) of NP and HA was 0.59 fM and 0.99 fM, and the coefficient of determination, R2, was 0.9998 and 0.9979, respectively. Two subtypes of influenza virus, A/Puerto Rico/8/1934 (H1N1) [PR8] and A/Panama/2007/99 (H3N2) [Pan99], were also quantified under established conditions, and the LOD of PR8 was 3.1 × 102 PFU/mL on targeting NP and 7.4 × 102 PFU/mL on targeting HA. The LOD of Pan99 was 5.3 × 102 PFU/mL on targeting NP. The specificity and robustness of the recombinant viral protein and influenza virus measurements using digital ELISA were also evaluated. Our measurement system showed enough specificity to discriminate the viral subtypes properly and showed sufficient inter- and intra-assay variations for both measurements of recombinant viral proteins and viruses, except for NP-targeting virus measurement.

Droplet-free digital immunoassay based on electrochemiluminescence

Here, we report a droplet-free digital immunoassay for goat immunoglobulin G (IgG) based on electrochemiluminescence (ECL) for simplified digital immunoassay. Digital enzyme-linked immunosorbent assay (ELISA) enabled ultrasensitive detection using digital counting immuno-reaction-induced femtoliter-sized droplets. However, the complicated preparation process of femtoliter-sized droplet assay limits the simplicity of digital ELISA. In this study, droplet-free digital ELISA using ECL was developed with digital counting ECL-induced beads instead of droplets along with a labeling reaction of immuno-reacted beads with ECL luminophore of [Ru(bpy)3]2+ in bulk solution. After antigen–antibody reaction on the beads using poly horseradish peroxidase (HRP) as a label, HRP reaction with [Ru(bpy)3]2+-conjugated tyramide was induced to modify the bead with [Ru(bpy)3]2+. The number of beads emitting ECL was counted on an indium tin oxide (ITO) electrode using tri-n-propylamine as a co-reactant. From result of poly HRP assay, our digital assay system required two poly HRP molecules on the bead to confirm ECL. Finally, a digital immunoassay was performed. The limit of detection for goat IgG was 0.7 pg/mL (4.7 fmol/L). Our simplified digital ELISA was less sensitive than conventional digital ELISA by 10-100-fold, however, we successfully performed digital ELISA based on ECL without complicated procedures for droplet array.

Development of a Fully Automated Desktop Analyzer and Ultrahigh Sensitivity Digital Immunoassay for SARS-CoV-2 Nucleocapsid Antigen Detection.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has had a significant impact on public health and the global economy. Several diagnostic tools are available for the detection of infectious diseases, with reverse transcription-polymerase chain reaction (RT-PCR) testing specifically recommended for viral RNA detection. However, this diagnostic method is costly, complex, and time-consuming. Although it does not have sufficient sensitivity, antigen detection by an immunoassay is an inexpensive and simpler alternative to RT-PCR. Here, we developed an ultrahigh sensitivity digital immunoassay (d-IA) for detecting SARS-CoV-2 nucleocapsid (N) protein as antigens using a fully automated desktop analyzer based on a digital enzyme-linked immunosorbent assay. Methods: We developed a fully automated d-IA desktop analyzer and measured the viral N protein as an antigen in nasopharyngeal (NP) swabs from patients with coronavirus disease. We studied nasopharyngeal swabs of 159 and 88 patients who were RT-PCR-negative and RT-PCR-positive, respectively. Results: The limit of detection of SARS-CoV-2 d-IA was 0.0043 pg/mL of N protein. The cutoff value was 0.029 pg/mL, with a negative RT-PCR distribution. The sensitivity of RT-PCR-positive specimens was estimated to be 94.3% (83/88). The assay time was 28 min. Conclusions: Our d-IA system, which includes a novel fully automated desktop analyzer, enabled detection of the SARS-CoV-2 N-protein with a comparable sensitivity to RT-PCR within 30 min. Thus, d-IA shows potential for SARS-CoV-2 detection across multiple diagnostic centers including small clinics, hospitals, airport quarantines, and clinical laboratories.

Highly Sensitive Serum Protein Analysis Using Magnetic Bead-Based Proximity Extension Assay.

Many protein biomarkers are present in biofluids at a very low level but may play critical roles in important biological processes. The fact that these low-abundance proteins remain largely unexplored underscores the importance of developing new tools for highly sensitive protein detection. Although digital enzyme-linked immunosorbent assay (ELISA) has demonstrated ultrahigh sensitivity compared with conventional ELISA, the requirement of specialized instruments limits the accessibility and prevents the widespread implementation. On the other hand, proximity ligation assays (PLA) and proximity extension assays (PEA) show sensitive and specific protein detection using regular laboratory setups, but their sensitivity needs to be further improved to match digital ELISA. To achieve highly sensitive protein detection with minimal accessibility limitation, we develop a magnetic bead-based PEA (magPEA), which posts triple epitope recognition requirement and enables extensive washing for improved sensitivity and enhanced specificity. We demonstrate that the incorporation of magnetic beads into PEA workflow facilitates orders of magnitude sensitivity improvement compared with conventional ELISA, homogeneous PEA, and solid-phase PLA and achieves limits of detection close to that of digital ELISA when using IL-6, IL-8, and GM-CSF as validation. Our magPEA provides a simple approach for highly sensitive protein detection that can be readily implemented to other laboratories and will thus ultimately accelerate the study of the low abundance protein biomarkers in the future.

No increase in inflammation in late-life major depression screened to exclude physical illness

Depression is a common and debilitating disorder in the elderly. Late-life depression (LLD) has been associated with inflammation and elevated levels of proinflammatory cytokines including interleukin (IL)-1β, tumor necrosis factor-alpha, and IL-6, but often depressed individuals have comorbid medical conditions that are associated with immune dysregulation. To determine whether depression has an association with inflammation independent of medical illness, 1120 adults were screened to identify individuals who had clinically significant depression but not medical conditions associated with systemic inflammation. In total, 66 patients with LLD screened to exclude medical conditions associated with inflammation were studied in detail along with 26 age-matched controls (HC). At baseline, circulating cytokines were low and similar in LLD and HC individuals. Furthermore, cytokines did not change significantly after treatment with either an antidepressant (escitalopram 20 mg/day) or an antidepressant plus a COX-2 inhibitor or placebo, even though depression scores improved in the non-placebo treatment arms. An analysis of cerebrospinal fluid in a subset of individuals for IL-1β using an ultrasensitive digital enzyme-linked immunosorbent assay revealed low levels in both LLD and HC at baseline. Our results indicate that depression by itself does not result in systemic or intrathecal elevations in cytokines and that celecoxib does not appear to have an adjunctive antidepressant role in older patients who do not have medical reasons for having inflammation. The negative finding for increased inflammation and the lack of a treatment effect for celecoxib in this carefully screened depressed population taken together with multiple positive results for inflammation in previous studies that did not screen out physical illness support a precision medicine approach to the treatment of depression that takes the medical causes for inflammation into account.

High-Throughput, High-Multiplex Digital Protein Detection with Attomolar Sensitivity.

A major challenge in many clinical diagnostic applications is the measurement of low-abundance proteins and other biomolecules in biological fluids. Digital technologies such as the digital enzyme-linked immunosorbent assay (ELISA) have enabled 1000-fold increases in sensitivity over conventional protein detection methods. However, current digital ELISA technologies still possess insufficient sensitivities for many rare protein biomarkers and require specialized instrumentation or time-consuming workflows that have limited their widespread implementation. To address these challenges, we have developed a more sensitive and streamlined digital ELISA platform, Molecular On-bead Signal Amplification for Individual Counting (MOSAIC), which attains low attomolar limits of detection, with an order of magnitude enhancement in sensitivity over these other methods. MOSAIC uses a rapid, automatable flow cytometric readout that vastly increases throughput and is easily integrated into existing laboratory infrastructure. As MOSAIC provides high sampling efficiencies for rare target molecules, assay bead number can readily be tuned to enhance signal-to-background with high measurement precision. Furthermore, the solution-based signal readout of MOSAIC expands the number of analytes that can simultaneously be measured for higher-order multiplexing with femtomolar sensitivities or below, compared with microwell- or droplet-based digital methods. As a proof of principle, we apply MOSAIC toward improving the detectability of low-abundance cytokines in saliva and ultrasensitive multiplexed measurements of eight protein analytes in plasma and saliva. The attomolar sensitivity, high throughput, and broad multiplexing abilities of MOSAIC provide highly accessible and versatile ultrasensitive capabilities that can potentially accelerate protein biomarker discovery and diagnostic testing for diverse disease applications.

Highly accurate multiprotein detection on a digital ELISA platform.

The emerging single-molecule detection platform digital enzyme-linked immunosorbent assay (ELISA) can detect numerous proteins simultaneously at serum concentrations as low as picograms per milliliter. We sought to improve cytokine detection with this platform to aid diagnosis of conditions such as allergy and asthma. We developed a multiple single-molecule detection digital ELISA approach, through the application of encoded magnetic microbeads to simultaneously detect three cytokines in one serum sample. We tested the approach's utility to distinguish asthma-related cytokines in children. Concentrations of interleukin-4 (IL-4) and IL-6 were significantly higher in children with asthma than in healthy controls, while the concentration of interferon-γ (IFN-γ) was significantly lower. Our method has higher accuracy than conventional methods, and our results indicate that the proposed improved high-sensitivity digital ELISA-based diagnosis approach can facilitate early detection and treatment of childhood asthma or related diseases.

Enzyme-based digital bioassay technology - key strategies and future perspectives.

Digital bioassays based on single-molecule enzyme reactions represent a new class of bioanalytical methods that enable the highly sensitive detection of biomolecules in a quantitative manner. Since the first reports of these methods in the 2000s, there has been significant growth in this new bioanalytical strategy. The principal strategy of this method is to compartmentalize target molecules in micron-sized reactors at the single-molecule level and count the number of microreactors showing positive signals originating from the target molecule. A representative application of digital bioassay is the digital enzyme-linked immunosorbent assay (ELISA). Owing to their versatility, various types of digital ELISAs have been actively developed. In addition, some disease markers and viruses possess catalytic activity, and digital bioassays for such enzymes and viruses have, thus, been developed. Currently, with the emergence of new microreactor technologies, the targets of this methodology are expanding from simple enzymes to more complex systems, such as membrane transporters and cell-free gene expression. In addition, multiplex or multiparametric digital bioassays have been developed to assess precisely the heterogeneities in sample molecules/systems that are obscured by ensemble measurements. In this review, we first introduce the basic concepts of digital bioassays and introduce a range of digital bioassays. Finally, we discuss the perspectives of new classes of digital bioassays and emerging fields based on digital bioassay technology.

Microchamber-Free Digital Flow Cytometric Analysis of T4 Polynucleotide Kinase Phosphatase Based on Single-Enzyme-to-Single-Bead Space-Confined Reaction.

Digital bioassays have attracted extensive attention in biomedical applications due to their ultrahigh sensitivity. However, traditional digital bioassays require numerous microchambers such as droplets or microwells, which restricts their application scope. Herein, we propose a microchamber-free flow cytometric method for the digital quantification of T4 polynucleotide kinase phosphatase (T4 PNKP) based on an unprecedented phenomenon that each T4 PNKP molecule-catalyzed reaction can be spatially self-confined on a single microbead, which ultimately enables the one-target-to-one-fluorescence-positive microbead digital signal transduction. The digital signal-readout mode can clearly detect T4 PNKP concentrations as low as 1.28 × 10-10 U/μL, making it most sensitive method to date. Significantly, T4 PNKP can be specifically distinguished from other phosphatases and nucleases in complex samples by digitally counting the fluorescence-positive microbeads, which cannot be realized by traditional bulk measurement-based methods. Taking advantage of the novel space-confined enzymatic feature of T4 PNKP, this digital mechanism can use T4 PNKP as the enzyme label to fabricate digital sensing systems toward various biomolecules such as digital enzyme-linked immunosorbent assay (ELISA). Therefore, this work not only enlarges the toolbox for high-sensitivity biomolecule detection but also opens new gates to fabricate next-generation digital assays.

The optimization of an ultra-sensitive single molecule assay for the multiplexed detection of SARS-CoV-2 antigens

Abstract Introduction/Objective SARS-CoV-2 antigens, including the nucleocapsid (N) protein, spike protein, and its S1 subunit have served as key biomarkers for research and diagnostic purposes. We previously developed quantitative single molecule array (Simoa) assays to measure the concentration of spike, S1 subunit and N protein in plasma samples with femtomolar limits of detection. We aimed to test antibodies that were not available early in the pandemic, reduce assay cross-reactivity, develop a multiplexed assay for spike, S1, and N protein in order to minimize the sample volume needed. Methods/Case Report Using the Simoa platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 17 S1 subunit and spike antibodies for a total of 130 antibody-pair combinations, we performed dilution linearity experiments to determine the ideal dilution factor, spike and recovery experiments, tested the assay using S1 subunit from other human coronavirus HKV1, NL63, and 229E, pre-pandemic plasma samples from patients that were sick with viral or bacterial respiratory infections. We then used the best antibody pairs to measure S1 and spike in plasma samples collected from patients with severe SARS-CoV-2. Lastly, we conjugated the best-performing capture antibodies for spike, S1 and N to beads labeled with different fluorophores to test if the assay for all three antigens could be multiplexed. Results (if a Case Study enter NA) We observed no cross-reactivity with S1 from other coronavirus strains, no detection of S1 or spike in a cohort of 30 pre-pandemic samples and successfully developed a multiplexed assay for the detection of spike, S1, and N protein, enabling us to use 50% less sample volume. Conclusion Reduction of necessary sample volume is important for studies involving multisystem inflammatory syndrome in children (MIS-C), and possible adverse effects of SARS-CoV-2 vaccinations on children and young adults. An improved assay with minimal cross-reactivity will also be useful to study individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).

Extracellular vesicles as distinct biomarker reservoirs for mild traumatic brain injury diagnosis.

Mild traumatic brain injury does not currently have a clear molecular diagnostic panel to either confirm the injury or to guide its treatment. Current biomarkers for traumatic brain injury rely mainly on detecting circulating proteins in blood that are associated with degenerating neurons, which are less common in mild traumatic brain injury, or with broad inflammatory cascades which are produced in multiple tissues and are thus not brain specific. To address this issue, we conducted an observational cohort study designed to measure a protein panel in two compartments—plasma and brain-derived extracellular vesicles—with the following hypotheses: (i) each compartment provides independent diagnostic information and (ii) algorithmically combining these compartments accurately classifies clinical mild traumatic brain injury. We evaluated this hypothesis using plasma samples from mild (Glasgow coma scale scores 13–15) traumatic brain injury patients (n = 47) and healthy and orthopaedic control subjects (n = 46) to evaluate biomarkers in brain-derived extracellular vesicles and plasma. We used our Track Etched Magnetic Nanopore technology to isolate brain-derived extracellular vesicles from plasma based on their expression of GluR2, combined with the ultrasensitive digital enzyme-linked immunosorbent assay technique, Single-Molecule Array. We quantified extracellular vesicle-packaged and plasma levels of biomarkers associated with two categories of traumatic brain injury pathology: neurodegeneration and neuronal/glial damage (ubiquitin C-terminal hydrolase L1, glial fibrillary acid protein, neurofilament light and Tau) and inflammation (interleukin-6, interleukin-10 and tumour necrosis factor alpha). We found that GluR2+ extracellular vesicles have distinct biomarker distributions than those present in the plasma. As a proof of concept, we showed that using a panel of biomarkers comprised of both plasma and GluR2+ extracellular vesicles, injured patients could be accurately classified versus non-injured patients.

Simultaneous detection of the spike and nucleocapsid proteins from SARS-CoV-2 based on ultrasensitive single molecule assays.

Nucleic acid detection technology based on polymerase chain reaction (PCR) and antibody detection based on immunochromatography still have many problems such as false negatives for the diagnosis of coronavirus disease 2019 (COVID-19). Therefore, it is of great importance to develop new techniques to improve the diagnostic accuracy of COVID-19. We herein developed an ultrasensitive, rapid, and duplex digital enzyme-linked immunosorbent assay (dELISA) for simultaneous detection of spike (S-RBD) and nucleocapsid (N) proteins of SARS-CoV-2 based on a single molecule array. This assay effectively combines magnetic bead encoding technology and the ultrasensitive detection capability of a single molecule array. The detection strategies of S-RBD protein and N-protein exhibited wide response ranges of 0.34–1065 pg/mL and 0.183–338 pg/mL with detection limits of 20.6 fg/mL and 69.8 fg/mL, respectively. It is a highly specific method for the simultaneous detection of S-RBD protein and N-protein and has minimal interference from other blood proteins. Moreover, the spike assay showed a satisfactory and reproducible recovery rate for the detection of S-RBD protein and N-protein in serum samples. Overall, this work provides a highly sensitive method for the simultaneous detection of S-RBD protein and N-protein, which shows ultrasensitivity and high signal-to-noise ratio and contributes to improve the diagnosis accuracy of COVID-19.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03435-z.

Single Molecule Assay for Ultrasensitive Detection of Cathepsin B in Human Blood.

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.

Rapid single-molecule digital detection of protein biomarkers for continuous monitoring of systemic immune disorders

Digital protein assays have great potential to advance immunodiagnostics because of their single-molecule sensitivity, high precision, and robust measurements. However, translating digital protein assays to acute clinical care has been challenging because it requires deployment of these assays with a rapid turnaround. Herein, we present a technology platform for ultrafast digital protein biomarker detection by using single-molecule counting of immune-complex formation events at an early, pre-equilibrium state. This method, which we term “pre-equilibrium digital enzyme-linked immunosorbent assay” (PEdELISA), can quantify a multiplexed panel of protein biomarkers in 10 µL of serum within an unprecedented assay incubation time of 15 to 300 seconds over a 104 dynamic range. PEdELISA allowed us to perform rapid monitoring of protein biomarkers in patients manifesting post-chimeric antigen receptor T-cell therapy cytokine release syndrome, with ∼30-minute sample-to-answer time and a sub–picograms per mL limit of detection. The rapid, sensitive, and low-input volume biomarker quantification enabled by PEdELISA is broadly applicable to timely monitoring of acute disease, potentially enabling more personalized treatment.