Antibody-coated Magnetic Beads Research Articles

Investigation and Demonstration for immunoassay technology based on impedance Amplifies of magnetic and dielectric microbeads

BackgroundBead-based immunoassays typically require researchers to perform the analysis in a laboratory setting, involving complex processing procedures, most of the time, constant potential or current instruments are used to measure the bio-impedance change. However, almost all biological materials are decorated on the electrode surface to obtain the maximum sensitivity, resulting in difficulty to clean, separate, and other applications of fixed bead-based immunoassay. ResultWe present an innovative unfixed bead-based immunoassay technology that utilizes electrical impedance characteristics, offering the advantages of being rapid, portable, miniaturized, and cost-effective. This technique which is known as Impedance-enhanced electrochemical immunoassay (IE-ECIA) employs antibody-coated Magnetic beads (MBs) and Dielectric beads (DBs) linked to antigens for quantitative with miniaturization electronic devices, which has been successfully applied in an immune impedance sensor for detecting the Dengue virus (DENV), which is an affordable test with a low-cost microfluidic impedance biosensor combined with immune magnetic and dielectric micro/nanobeads for antigen detection. HighlightThe proposed immunoassay technology introduces a novel microfluidic platform incorporating magnetic and dielectric beads alongside with an electrochemical impedance biosensor, therefore facilitating continuous-fluid quantitative analysis. This innovative and unconventional approach leverages free-motion multiplex beads housing magnetic and dielectric bead-bound antigens within a simple linear microchannel of microfluidic chip architecture, making the high-through measurements more efficient, enhanced Electrochemical immunoassay (ECIA) signals of Bio-on-magnetic-beads (BOMB). This advancement holds significant potential for enhancing diagnostic capabilities for emerging infectious diseases within clinical environments, thereby bolstering rapid detection capabilities and aiding in the advancement of novel pharmaceuticals, benefiting all the stakeholders.

Cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of Shigella flexneri.

Pathogen infections including Shigella flexneri have posed a significant threat to human health for numerous years. Although culturing and qPCR were the gold standards for pathogen detection, time-consuming and instrument-dependent restrict their application in rapid diagnosis and economically less-developed regions. Thus, it is urgently needed to develop rapid, simple, sensitive, accurate, and low-cost detection methods for pathogen detection. In this study, an immunomagnetic beads-recombinase polymerase amplification-CRISPR/Cas12a (IMB-RPA-CRISPR/Cas12a) method was built based on a cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of S. flexneri in the laboratory. Firstly, S. flexneri was specifically captured and enriched by IMB (Shigella antibody-coated magnetic beads), and the genomic DNA was released and used as the template in the RPA reaction. Then, the RPA products were mixed with the pre-loaded CRISPR/Cas12a for fluorescence visualization. The results were observed by naked eyes under LED blue light, with a sensitivity of 5CFU/mL in a time of 70min. With no specialized equipment or complicated technical requirements, the IMB-RPA-CRISPR/Cas12a diagnostic method can be used for visual, rapid, and simple detection of S. flexneri and can be easily adapted to monitoring other pathogens.

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk. hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function. hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

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.

Isolation of yeast mitochondria by affinity purification using magnetic beads.

Isolated mitochondria have been widely utilized in various model organisms to investigate the diverse functions of the organelle. Techniques such as differential centrifugation, density gradient ultracentrifugation and antibody-coated magnetic beads are employed for isolation of the organelle from whole cells. However, mitochondria isolated using differential centrifugation are often contaminated with other organelles; isolation using density gradient ultracentrifugation can reduce contamination but is time-intensive and requires large amounts of starting materials; and mitochondria isolated using antibody-coated magnetic beads are irreversibly bound to the beads. Here, we provide a step-by-step protocol for the isolation of highly pure mitochondria from Saccharomyces cerevisiae using a magnetic bead affinity purification method that overcomes these limitations. This protocol describes how to isolate mitochondria, tagged by insertion of 6 histidines (6xHis) into the chromosomal copy of the TOM70 (Translocase of outer membrane 70) gene using Ni-NTA (nickel(II) nitrilotriacetic acid) paramagnetic beads, and the subsequent release of mitochondria from the beads using a buffer containing imidazole. We provide examples of expected results, highlighting the purity, integrity and import activity of isolated mitochondria. These affinity-purified mitochondria are intact and functional, containing less contamination with cytosol and other organelles compared to mitochondria isolated by other methods. Our method is adaptable and can be applied to other model organisms that can be genetically manipulated using CRISPR or other methods.

Elastic Microstructures: Combining Biochemical, Mechanical, and Topographical Cues for the Effective Activation and Proliferation of Cytotoxic T Cells.

The ex vivo activation and proliferation of cytotoxic T cells are critical steps in adoptive immunotherapy. Today, T cells are activated by stimulation with antibody-coated magnetic beads, traditionally used for cell separation. Yet, efficient and controlled activation and proliferation of T cells require new antibody-bearing materials, which, in particular, deliver mechanical and topographic cues sensed by T cells. Here, we demonstrate a new approach for the activation and proliferation of human cytotoxic T cells using an elastic microbrush coated with activating and costimulatory antibodies. We found that the microbrush topography affects the protrusion of the cell membrane and the elastic response to the forces applied by cells and can be optimized to yield the strongest activation of T cells. In particular, T cells stimulated by a microbrush showed a three-fold increase in degranulation and release of cytokines over T cells stimulated with state-of-the-art magnetic beads. Furthermore, the microbrush induced a T-cell proliferation of T cells that was more prolonged and yielded much higher cell doubling than that done by the state-of-the-art methods. Our study provides an essential insight into the physical mechanism of T-cell activation and proliferation and opens the floodgates for the design of novel stimulatory materials for T-cell-based immunotherapy.

Alternating Magnetic Field-Promoted Nanoparticle Mixing: The On-Chip Immunocapture of Serum Neuronal Exosomes for Parkinson's Disease Diagnostics.

The analysis of cargo proteins in exosome subpopulations has considerable value in diagnostics but a translatable impact has been limited by lengthy or complex exosome extraction protocols. We describe herein a scalable, fast, and low-cost exosome extraction using an alternating (AC) magnetic field to support the dynamic mixing of antibody-coated magnetic beads (MBs) with serum samples within 3D-printed microfluidic chips. Zwitterionic polymer-coated MBs are, specifically, magnetically agitated and support ultraclean exosome capture efficiencies >70% from <50 μL of neat serum in 30 min. Applied herein to the immunocapture of neuronal exosomes using anti-L1CAM antibodies, prior to the array-based assaying of α-synuclein (α-syn) content by a standard duplex electrochemical sandwich ELISA, sub pg/mL detection was possible with an excellent coefficient of variation and a sample-to-answer time of ∼75 min. The high performance and semiautomation of this approach hold promise in underpinning low-cost Parkinson's disease diagnostics and is of value in exosomal biomarker analyses more generally.

Immunomagnetic-bead enriched culturomics (IMBEC) for isolating pathobionts from feces of colorectal cancer patients.

Culturomics employs various cultivating conditions to obtain different types of bacteria and new species. However, current culturomics lacks a highly efficient method for isolating specific pathobionts. Immunomagnetic bead technology, which uses magnetic beads conjugated with antibodies for capturing the antigen to realize enrichment of the targets, has been employed as an alternative method. In this study, we developed a novel method, immunomagnetic bead-enriched culturomics (IMBEC), in which magnetic bead-conjugated antibodies purified from the fecal samples of patients with colorectal cancer (CRC) were used to enrich and isolate potential pathobionts. A protocol for enriching potential pathobionts via immunomagnetic capture was developed by optimizing the concentrations of coupling reagents, NaCl, and detergent. The efficacy of pathobiont enrichment was compared between antibody-coated magnetic beads (antibody group) and nonconjugated blank magnetic beads (blank group). To determine the proinflammatory potential of isolates from both groups, we investigated their ability to induce cytokine production in THP-1 macrophages. This protocol was employed for isolating bacteria from 10 fecal samples of patients with CRC, which were simultaneously compared with those isolated from the blank group. A total of 209 bacterial species were isolated from both groups, including 173 from the antibody group, 160 from the blank group, and 124 from both groups. Bacteria isolated from the antibody group produced more proinflammatory cytokines than those isolated from the blank group. IMBEC is a promising method for relatively specific isolation of potential pathobionts for a particular disease of interest.

Isolation of the B Cell Immune Synapse for Proteomic Analysis.

Recent technical developments have fueled increasing utilization of proteomics to gain new insights into various aspects of cellular behavior. In this chapter, we describe a method to specifically isolate immune synapses from mouse primary B cells. The method utilizes antibody-coated magnetic beads to induce the formation of the immune synapses and describes a protocol for the extraction of the cell-bead adhesions for mass spectrometry analysis. Finally, this method enables unveiling the large-scale protein content of the immune synapse.

Microfluidic Magnetic Spatial Confinement Strategy for the Enrichment and Ultrasensitive Detection of MCF-7 and Escherichia coli O157:H7.

A microfluidic magnetic spatial confinement strategy was developed and employed to realize an ultrasensitive cell immunoassay. The straight confined channels in poly(dimethylsiloxane)-glass hybrid microchips were used as the enrichment and detection chambers for the proposed microfluidic magnetic cell immunoassays (μMCI). To accomplish the μMCI, prepared magnetic cell immunocomplexes were introduced into microchannels and preconcentrated in the detection zone under a permanent magnet. The magnetic cell immunocomplexes were constructed from aptamer-/antibody-coated magnetic beads and antibody-linked horseradish peroxidase-labeled target cells to guarantee the specificity and enhance the detection signal generated from the enzyme reaction. The sensitivity enhancement of μMCI was confirmed in a one-dimensional space confined microchamber, especially in the analysis of cells having more enzyme conjugating sites on their surface. This spatial confinement strategy based μMCI was then applied for model cell detection in the microchannel, the limits of detection (LODs) were 2 cells/mL for MCF-7 and 34 colony-forming unit/mL for Escherichia coli O157:H7 (E. coli O157:H7), which corresponded to up to 1202-fold LOD sensitivity improvement compared to the results of the similar immunoassays in microwell plates. The satisfactory selectivity and reproducibility of the strategy were also obtained. Moreover, it enabled rare MCF-7 detection in whole blood and E. coli O157:H7 detection in milk after time-shortened incubation. Constructing an appropriate confined space, this strategy can be extended to detect various cells with higher sensitivity, which provides a valuable approach for rare cell detection in practical applications.

ODP010 Relaxin Receptor RXFP1 Knockout in Adipose Tissue Progenitor Cells Results in Resistance to High Fat Diet-Induced Weigh Gain

Abstract Relaxin is a polypeptide hormone that signals through its cognate receptor RXFP1. Previous work demonstrated that global RXFP1-knockout mice developed age-related adipose tissue fibrosis and dysfunction. Our preliminary data suggested that adipose tissue progenitor cells (APC) express RXFP1 in the white adipose tissue in mice. To determine the role of APC in relaxin signaling, we crossed floxed RXFP1 mice (RXFP1-fl/fl) with mice expressing tamoxifen-inducible CRE recombinase under control of the platelet-derived growth factor receptor-β (PDGFRβ) promoter to produce conditional RXFP1 knockout in adipose tissue progenitor cells and fibroblasts (RXFP1-PKO). After tamoxifen treatment, both RXFP1-fl/fl and RXFP1-PKO mice were placed on a high fat diet (58% calories from fat) supplemented with sucrose and fructose in the drinking water, or chow diet for 16 weeks. At the end of the study, body mass composition was measured using an Echo-MRI instrument. Adipose tissue fibrosis was assessed histologically by Sirius red staining. In response to the HFD, the control (fl/fl) mice reached an average weight of 48.3 ± 0.6g, while the RXFP1-PKO mice were resistant to weight gain, reaching only 40.3 ±3g (p&amp;lt;. 05). The body mass composition changed in the RXFP1-fl/fl mice on HFD (34. 0% fat/55.2% lean) compared to chow (14.7% fat/68.9% lean). The RXFP1-PKO mice had a significantly lower gain in fat mass on HFD (29.7% fat/59.1% lean on HFD; 12.6% fat/71. 0% lean on chow) compared to the RXFP-fl/fl mice. Histological analysis of the inguinal white adipose tissue (iWAT) revealed that while RXFP1-fl/fl mice developed adipocyte hypertrophy in response to the HFD, the RXFP1-PKO mice had smaller adipocyte size. This was accompanied by increased pericellular and perivascular fibrosis in the iWAT as determined by Sirius red staining. To determine if differentiation of APC was altered by loss of RXFP1, the stromal-vascular fraction of iWAT was extracted by collagenase treatment, and the APC isolated using antibody-coated magnetic beads (Miltenyi Biotec). The purified cells were cultured to confluence, treated with or without tamoxifen to induce CRE expression, then treated with differentiation cocktail. The APC from the RXFP1-PKO mice had a marked impairment in the ability differentiate to mature adipocytes after treatment with tamoxifen compared to untreated cells. In summary, the loss of RXFP1 in PDGFRβ-expressing cells resulted in resistance to HFD-induced weight and adipose tissue mass gain. This was accompanied by increased fibrosis in the iWAT and decreased adipocyte size. The APC had decreased adipogenic potential after loss of RXFP1. Taken together, the findings support a role for RXFP1 in the differentiation of adipose tissue progenitor cells, and in adipose tissue expansion after exposure to an obesogenic diet. Presentation: No date and time listed

Cross-reactivity of antibodies to different rumen methanogens demonstrated using immunomagnetic capture technology.

Methane is produced in the rumen of ruminant livestock by methanogens, accounting for approximately 14.5% of anthropogenic greenhouse gas emissions in terms of global warming potential. The rumen contains a diversity of methanogens species, and only a few of these have been cultured. Immunomagnetic capture technology (ICT) is a simple and effective method to capture and concentrate target organisms in samples containing complex microflora. We hypothesized that antibody-coated magnetic beads could be used to demonstrate antibody specificity and cross-reactivity to methanogens in rumen samples. Sheep polyclonal antibodies raised against four isolates of rumen dwelling methanogens, Methanobrevibacter ruminantium strain M1, Methanobrevibacter sp. AbM4, Methanobrevibacter sp. D5, and Methanobrevibacter sp. SM9 or an equal mix of all four isolates, were used to coat paramagnetic beads. ICT was used together with flow cytometry and qPCR to optimize key parameters: the ratio of antibody to beads, coupling time between antibody and paramagnetic beads to produce immunomagnetic beads (IMBs), and optimal incubation time for the capture of methanogen cells by IMBs. Under optimized conditions, IMBs bound strongly to their respective isolates and showed a degree of cross-reactivity with isolates of other Methanobrevibacter spp. in buffer and in rumen fluid, and with resident methanogens in rumen content samples. The evidence provided here indicates that this method can be used to study the interaction of antibodies with antigens of rumen methanogens, to understand antigen cross-reactivity and antibody binding efficiency for the evaluation of antigens used for the development of a broad-spectrum anti-methanogen vaccine for the abatement of methane production.

Development of a Specific Anti-capsid Antibody- and Magnetic Bead-Based Immunoassay to Detect Human Norovirus Particles in Stool Samples and Spiked Mussels via Flow Cytometry.

Human noroviruses impose a considerable health burden globally. Here, a flow cytometry approach designed for their detection in biological waste and food samples was developed using antibody-coated magnetic beads. Antipeptide antibodies against murine norovirus and various human norovirus genotypes were generated for capture and coated onto magnetic beads. A flow cytometry assay was then implemented to detect bead-bound human norovirus GI.3 in patient stool samples and in norovirus-spiked mussel digestive tissues. The detection limit for stool samples was 105gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was ten-fold higher than in stool samples. Further assays showed a decrease in fluorescence intensity for heat- or UV-inactivated virus particles. Overall, we demonstrate the application of a flow cytometry approach for direct detection of small non-enveloped virus particles such as noroviruses. An adaptation of the technology to routine diagnostics has the potential to contribute a rapid and sensitive tool to norovirus outbreak investigations. Further improvements to the method, notably decreasing the detection limit of the approach, may allow the analysis of naturally contaminated food and environmental samples.

Isolation of primary human B lymphocytes from tonsils compared to blood as alternative source for ex vivo application

B lymphocytes (‘B cells’) are components of the human immune system with obvious potential for medical and biotechnological applications. Here, we discuss the isolation of primary human B cells from both juvenile and adult tonsillar material using a two-step procedure based on gradient centrifugation followed by separation on a nylon wool column as alternative to the current gold standard, i.e., negative immunosorting from buffy coats by antibody-coated magnetic beads. We show that the nylon wool separation is a low-cost method well suited to the isolation of large amounts of primary B cells reaching purities ≥ 80%. More importantly, this method allows the preservation of all B cell subsets, while MACS sorting seems to be biased against a certain B cell subtype, namely the CD27+ B cells. Importantly, compared to blood, the excellent recovery yield during purification of tonsillar B cells provides high number of cells, hence increases the number of subsequent experiments feasible with identical cell material, consequently improving comparability of results. The cultivability of the isolated B cells was demonstrated using pokeweed mitogen (PWM) as a stimulatory substance. Our results showed for the first time that the proliferative response of tonsillar B cells to mitogens declines with the age of the donor. Furthermore, we observed that PWM treatment stimulates the proliferation of a dedicated subpopulation and induces some terminal differentiation with ASCs signatures. Taken together this indicates that the proposed isolation procedure preserves the proliferative capability as well as the differentiation capacity of the B cells.

An integrated magneto-electrochemical device for the rapid profiling of tumour extracellular vesicles from blood plasma.

The analysis of proteins expressed on circulating extracellular vesicles (EVs) could facilitate the diagnosis of different types of cancers. EV assays however have lengthy sample workups and limited throughput and sensitivity, making them unsuitable for routine clinical use. Here, we report a high-throughput assay that integrates EV enrichment, via antibody-coated magnetic beads, with the detection of EV-bound antibodies, via an electrochemical reaction. The assay requires less than one hour, is performed on plasma samples, and its 96-well plate format enables measurements in parallel via a prototype reader. Using samples from patients with colorectal cancer or healthy volunteers, we identified a panel of biomarkers (EGFR, EpCAM, CD24, GPA33) in circulating EVs that, when combined, showed higher diagnostic accuracy (>96%) than conventional assays. In a prospective cohort, the combined biomarker profile enabled assigning patients to a high- or a low-risk 5-year disease-free survival group, and the serial monitoring of EVs during therapy showed values declined after surgery yet increased upon relapse. Biomarker panels from plasma EVs may be suitable for the non-invasive monitoring of disease trajectory.

Functionally Active Eosinophil Purification from Peripheral Blood.

The choice of isolation technique for human peripheral blood eosinophils contributes to the understanding of clinically relevant data derived from in vitro research. Since the 1990s, eosinophils have been conventionally isolated via density gradient centrifugation followed by negative immunomagnetic selection using anti-CD16 antibody-coated magnetic beads. Due to recent advancements in molecular techniques, "newer" methods have been made commercially available that drastically reduce user handling and processing time while maintaining high population purity. Here, we describe an isolation procedure using one of these methods, the human MACSxpress® Whole Blood Isolation Kit, as well as outline protocols for differential staining and flow cytometry analysis to evaluate the purity and activation state of isolated cells. In addition, we highlight an in vitro degranulation assay that may be used to verify the intact functionality of the isolated eosinophils.

Abstract 773: Tumor-derived Extracellular Vesicles (EVs) expressing TGFb as potential biomarkers for anti-TGFb antibody activity and drug activity in liquid biopsy

Abstract The field of liquid biopsies is of enormous interest in cancer using a noninvasive process. There are currently three major approaches: Circulating Cell Tumors (CTCs), circulating cell free DNA (cfDNA) and Extracellular Vesicles (EVs) such as exosomes, microvesicles and apoptotic bodies. The EVs are cell-derived small membrane vesicles secreted by cells that convey biological messages, either by surface-to-surface interaction or by shuttling bioactive molecules to a recipient cell's cytoplasm. The EVs released by cells harbor the cargos of their original cells and could be a good tool to follow drug activity. We are developing anti-TGFβ antibody SAR439459 for the treatment of cancer (NCT03192345), and in this vitro study we explore the effect of anti-TGFβ antibody in EVs carrying TGFβ. The cargo of EVs were analyzed by an Elisa test for both latent and active free TGFβ and by miRNA expression. Cell lines were selected based on their expression of TGFβ (A549, DU45, PC3, MCF7 and Caco2), treated for 72h and compared with respective untreated controls. The EVs were isolated from the supernatant and purified. Nanoparticle Tracking Analysis was used to quantify and determine size distribution of EVs. The expression of active free and latent TGFβ was done using Elisa tests. TGFβ antibody-coated magnetic beads were used to capture EVs, and miRNA expression sequencing was done using the Illumina NextSeq. Anti-TGFβ treatment reduced notably the expression of latent TGFβ in the supernatant of cultured tumor cells; the active free TGFβ was very low expressed or not detected in the supernatants. EVs express both latent and active free TGFβ; the anti-TGFβ antibody treatment significantly reduced the active free TGFβ and altered miRNA expression in EVs. Finally, using TGFβ Antibody-coated magnetic beads, we demonstrate that intact EVs express TGFβ at their surface. In conclusion, these results suggest that EVs express TGFβ at their surface and carry the active free TGFβ. Finally, the expression of active free TGFβ in EVs is decreased with anti-TGFβ antibody, suggesting that it might be a biomarker for monitoring anti-TGFβ antibody activity in patients by liquid biopsies. Citation Format: Manoel Nunes, Camille Bernard, Gaelle Muzard, Wilson Dos-Santos-Bele, Jack Pollard, Alexei Protopopov, Colette Dib. Tumor-derived Extracellular Vesicles (EVs) expressing TGFb as potential biomarkers for anti-TGFb antibody activity and drug activity in liquid biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 773.

Detection of three tumor biomarkers in human lung cancer serum using single particle inductively coupled plasma mass spectrometry combined with magnetic immunoassay

A new method for simultaneously determining cytokeratin fragment antigen 21–1 (CYFRA21-1), carcinoembryonic antigen (CEA), and carbohydrate antigen (CA15–3) was developed using single-particle, ICP-MS-based, magnetic immunoassay. Gold nanoparticles (AuNPs), ZnSe quantum dots (QDs), and Silver nanoparticles (AgNPs) were employed to tag antibodies. A highly efficient capture of CYFRA21-1, CEA, and CA15–3 was achieved using biotinylated, antibody-coated magnetic beads. AuNPs/ZnSe QDs/AgNPs-tagged second antibodies helped realize sensitive quantification of the target antigen using single particle ICP-MS. Under optimum conditions, CYFRA21-1, CEA, and CA15–3 linear concentration ranges were 0.05–50 ng mL−1, 0.02–100 ng mL−1, and 0.6–250 mU mL−1. The correlation coefficients were 0.9999, 0.9987, and 0.9970, respectively. CYFRA21-1, CEA, and CA15–3 detection limits were 0.02 ng mL−1, 0.006 ng mL−1, and 0.25 mU mL−1, respectively. Relative standard deviations (RSD) for CYFRA21-1, CEA, and CA15–3 were 3.2%, 4.4%, and 4.2%, respectively. The method simultaneously detected CYFRA21-1, CEA, and CA15–3 in human lung cancer serum samples with recoveries of between 89.8% and 101%.

Development of a multiplex fully automated assay for rapid quantification of CD4+ T cells from whole blood

The development of cost-effective and rapid assays for the accurate counting of CD4 cells has remained prime focus for disease management. The lack of such assays has severely affected people living in resource-limited disease prevalent areas. CD4 count information plays a vital role in the effective management of HIV disease. There is an unmet need to develop rapid, cost-effective, portable and user-friendly point-of-care (POC) disease diagnostic platform technology for CD4+ T cell counting. Here, we have developed a flow-free magnetic actuation platform that uses antibody-coated magnetic beads to efficiently capture CD4+ T cells from a 30 μL drop of whole blood. On-chip cell lysate electrical impedance spectroscopy has been utilized to quantify the isolated CD4 cells. The developed assay has a limit of detection of 25 cells per μL and provides accurate CD4 counts in the range of 25–800 cells per μL. The whole immunoassay along with the enumeration process is very rapid and provides CD4 quantification results within 5 min time frame. The assay does not require off-chip sample preparation steps and minimizes human involvement to a greater extent. The developed impedance-based immunoassay has potential to significantly improve the CD4 enumeration process especially for POC settings.

Placental Syncytiotrophoblast-Derived Extracellular Vesicles Carry Active NEP (Neprilysin) and Are Increased in Preeclampsia.

NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a variety of peptides including vasodilators, natriuretics, and diuretics. Higher levels of NEP result in hypertension-a cardinal feature of the placental disease preeclampsia. Syncytiotrophoblast-derived extracellular vesicles (EVs), comprising microvesicles and exosomes, are released into the peripheral circulation in pregnancy and are postulated as a key mechanism coupling placental dysfunction and maternal phenotype in preeclampsia. We aimed to determine whether higher levels of active NEP are found in syncytiotrophoblast-derived EVs in preeclampsia compared with normal pregnancy. Using immunostaining and Western blotting, we first demonstrated that NEP levels are greater not only in preeclampsia placental tissue but also in syncytiotrophoblast-derived microvesicles and exosomes isolated from preeclampsia placentas ( P<0.05, n=5). We confirmed placental origin using antibody-coated magnetic beads to isolate NEP-bound vesicles, finding that they stain for placental alkaline phosphatase. NEP on syncytiotrophoblast-derived EVs is active and inhibited by thiorphan ( P<0.01, n=3; specific inhibitor). Syncytiotrophoblast-derived microvesicles, isolated from peripheral plasma, demonstrated higher NEP expression in preeclampsia using flow cytometry ( P<0.05, n=8). We isolated plasma exosomes using size-exclusion chromatography and showed greater NEP activity in preeclampsia ( P<0.05, n=8). These findings show that the placenta releases active NEP into the maternal circulation on syncytiotrophoblast-derived EVs, at significantly greater levels in preeclampsia. NEP has pathological roles in hypertension, heart failure, and amyloid deposition, all of which are features of preeclampsia. Circulating syncytiotrophoblast-derived EV-bound NEP thus may contribute to the pathogenesis of this disease.