RESUMO
Local immune activation at mucosal surfaces, mediated by mucosal lymphoid tissues, is vital for effective immune responses against pathogens. While pathogens like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can spread to multiple organs, patients with coronavirus disease 2019 (COVID-19) primarily experience inflammation and damage in their lungs. To investigate this apparent organ-specific immune response, we develop an analytical framework that recognizes the significance of mucosal lymphoid tissues. This framework combines histology, immunofluorescence, spatial transcript profiling, and mathematical modeling to identify cellular and gene expression differences between the lymphoid tissues of the lung and the gut and predict the determinants of those differences. Our findings indicate that mucosal lymphoid tissues are pivotal in organ-specific immune response to SARS-CoV-2, mediating local inflammation and tissue damage and contributing to immune dysfunction. The framework developed here has potential utility in the study of long COVID and may streamline biomarker discovery and treatment design for diseases with differential pathologies at the organ level.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Síndrome de COVID-19 Pós-Aguda , Inflamação , ImunidadeRESUMO
The discovery and characterization of antigen-specific CD8+ T cell clonotypes typically involves the labor-intensive synthesis and construction of peptide-MHC tetramers. We adapt single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation, showing that hundreds can be rapidly prepared across multiple Class I HLA alleles. We use this platform to explore the impact of peptide and SCT template mutations on protein expression yield, thermal stability, and functionality. SCT libraries were an efficient tool for identifying T cells recognizing commonly reported viral epitopes. We then construct SCT libraries to capture SARS-CoV-2 specific CD8+ T cells from COVID-19 participants and healthy donors. The immunogenicity of these epitopes is validated by functional assays of T cells with cloned TCRs captured using SCT libraries. These technologies should enable the rapid analyses of peptide-based T cell responses across several contexts, including autoimmunity, cancer, or infectious disease.
Assuntos
Linfócitos T CD8-Positivos , COVID-19 , Humanos , SARS-CoV-2/genética , Antígenos , Epitopos , Peptídeos/genéticaRESUMO
T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Edição de Genes , Neoplasias , Medicina de Precisão , Receptores de Antígenos de Linfócitos T , Linfócitos T , Transgenes , Humanos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Biópsia , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Síndrome da Liberação de Citocina/complicações , Progressão da Doença , Encefalite/complicações , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Mutação , Neoplasias/complicações , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , Segurança do Paciente , Medicina de Precisão/efeitos adversos , Medicina de Precisão/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transgenes/genética , Antígenos HLA/imunologia , Sistemas CRISPR-CasRESUMO
The response of patients with recurrent glioblastoma multiforme to neoadjuvant immune checkpoint blockade has been challenging to interpret due to the inter-patient and intra-tumor heterogeneity. We report on a comparative analysis of tumor tissues collected from patients with recurrent glioblastoma and high-risk melanoma, both treated with neoadjuvant checkpoint blockade. We develop a framework that uses multiplex spatial protein profiling, machine learning-based image analysis, and data-driven computational models to investigate the pathophysiological and molecular factors within the tumor microenvironment that influence treatment response. Using melanoma to guide the interpretation of glioblastoma analyses, we interrogate the protein expression in microscopic compartments of tumors, and determine the correlates of cytotoxic CD8+ T cells, tumor growth, treatment response, and immune cell-cell interaction. This work reveals similarities shared between glioblastoma and melanoma, immunosuppressive factors that are unique to the glioblastoma microenvironment, and potential co-targets for enhancing the efficacy of neoadjuvant immune checkpoint blockade.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Antígeno CTLA-4/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Inibidores de Checkpoint Imunológico/uso terapêutico , Melanoma/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Adulto , Idoso , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/análise , Neoplasias Encefálicas/patologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Glioblastoma/patologia , Humanos , Ipilimumab/uso terapêutico , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Nivolumabe/uso terapêutico , Resultado do Tratamento , Microambiente Tumoral/imunologiaRESUMO
Many approaches to identify therapeutically relevant neoantigens couple tumor sequencing with bioinformatic algorithms and inferred rules of tumor epitope immunogenicity. However, there are no reference data to compare these approaches, and the parameters governing tumor epitope immunogenicity remain unclear. Here, we assembled a global consortium wherein each participant predicted immunogenic epitopes from shared tumor sequencing data. 608 epitopes were subsequently assessed for T cell binding in patient-matched samples. By integrating peptide features associated with presentation and recognition, we developed a model of tumor epitope immunogenicity that filtered out 98% of non-immunogenic peptides with a precision above 0.70. Pipelines prioritizing model features had superior performance, and pipeline alterations leveraging them improved prediction performance. These findings were validated in an independent cohort of 310 epitopes prioritized from tumor sequencing data and assessed for T cell binding. This data resource enables identification of parameters underlying effective anti-tumor immunity and is available to the research community.
Assuntos
Antígenos de Neoplasias/imunologia , Epitopos/imunologia , Neoplasias/imunologia , Alelos , Apresentação de Antígeno/imunologia , Estudos de Coortes , Humanos , Peptídeos/imunologia , Receptor de Morte Celular Programada 1 , Reprodutibilidade dos TestesRESUMO
Non-invasively probing metabolites within single live cells is highly desired but challenging. Here we utilize Raman spectro-microscopy for spatial mapping of metabolites within single cells, with the specific goal of identifying druggable metabolic susceptibilities from a series of patient-derived melanoma cell lines. Each cell line represents a different characteristic level of cancer cell de-differentiation. First, with Raman spectroscopy, followed by stimulated Raman scattering (SRS) microscopy and transcriptomics analysis, we identify the fatty acid synthesis pathway as a druggable susceptibility for differentiated melanocytic cells. We then utilize hyperspectral-SRS imaging of intracellular lipid droplets to identify a previously unknown susceptibility of lipid mono-unsaturation within de-differentiated mesenchymal cells with innate resistance to BRAF inhibition. Drugging this target leads to cellular apoptosis accompanied by the formation of phase-separated intracellular membrane domains. The integration of subcellular Raman spectro-microscopy with lipidomics and transcriptomics suggests possible lipid regulatory mechanisms underlying this pharmacological treatment. Our method should provide a general approach in spatially-resolved single cell metabolomics studies.
Assuntos
Melanoma/metabolismo , Metabolômica/métodos , Microscopia/métodos , Análise Espectral Raman/métodos , Apoptose , Linhagem Celular Tumoral , Ácidos Graxos/metabolismo , Humanos , Gotículas Lipídicas , Metabolismo dos Lipídeos , Lipidômica , Lipídeos , Ácido Oleico , Estearoil-CoA Dessaturase/metabolismo , TranscriptomaRESUMO
T cells can be controllably stimulated through antigen-specific or nonspecific protocols. Accompanying functional hallmarks of T cell activation can include cytoskeletal reorganization, cell size increase, and cytokine secretion. Photon-induced near-field electron microscopy (PINEM) is used to image and quantify evanescent electric fields at the surface of T cells as a function of various stimulation conditions. While PINEM signal strength scales with multiple of the biophysical changes associated with T cell functional activation, it mostly strongly correlates with antigen-engagement of the T cell receptors, even under conditions that do not lead to functional T cell activation. PINEM image analysis suggests that a stimulation-induced reorganization of T cell surface structure, especially over length scales of a few hundred nanometers, is the dominant contributor to these PINEM signal changes. These experiments reveal that PINEM can provide a sensitive label-free probe of nanoscale cellular surface structures.
Assuntos
Ativação Linfocitária , Linfócitos T/ultraestrutura , Humanos , Células Jurkat , Microscopia Eletrônica/métodos , Propriedades de SuperfícieRESUMO
Adaptive immunity is based on peptide antigen recognition. Our ability to harness the immune system for therapeutic gain relies on the discovery of the T cell receptor (TCR) genes that selectively target antigens from infections, mutated proteins, and foreign agents. Here we present a method that selectively labels peptide antigen-specific CD8+ T cells using magnetic nanoparticles functionalized with peptide-MHC tetramers, isolates these specific cells within an integrated microfluidic device, and directly amplifies the TCR genes for sequencing. Critically, the identity of the peptide recognized by the TCR is preserved, providing the link between peptide and gene. The platform requires inputs on the order of just 100 000 CD8+ T cells, can be multiplexed for simultaneous analysis of multiple peptides, and performs sorting and isolation on chip. We demonstrate 1000-fold sensitivity enhancement of detecting antigen-specific TCRs relative to bulk analysis and simultaneous capture of two virus antigen-specific TCRs from a population of T cells.
Assuntos
Antígenos/genética , Técnicas Analíticas Microfluídicas , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T CD8-Positivos , Células Cultivadas , Humanos , Nanopartículas de Magnetita/química , Técnicas Analíticas Microfluídicas/instrumentação , Tamanho da Partícula , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines.
Assuntos
Antígenos de Neoplasias/sangue , Melanoma/sangue , Melanoma/imunologia , Linfócitos T/imunologia , Biópsia , Células HEK293 , Humanos , Imunoterapia , Células Jurkat , Cinética , Linfócitos do Interstício Tumoral/imunologia , Nanopartículas de Magnetita/química , Complexo Principal de Histocompatibilidade , Melanoma/patologia , Melanoma/secundário , Ácidos Nucleicos/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Reprodutibilidade dos Testes , Tomografia Computadorizada por Raios XRESUMO
Phenotypic plasticity is associated with non-genetic drug tolerance in several cancers. Such plasticity can arise from chromatin remodeling, transcriptomic reprogramming, and/or protein signaling rewiring, and is characterized as a cell state transition in response to molecular or physical perturbations. This, in turn, can confound interpretations of drug responses and resistance development. Using BRAF-mutant melanoma cell lines as the prototype, we report on a joint theoretical and experimental investigation of the cell-state transition dynamics associated with BRAF inhibitor drug tolerance. Thermodynamically motivated surprisal analysis of transcriptome data was used to treat the cell population as an entropy maximizing system under the influence of time-dependent constraints. This permits the extraction of an epigenetic potential landscape for drug-induced phenotypic evolution. Single-cell flow cytometry data of the same system were modeled with a modified Fokker-Planck-type kinetic model. The two approaches yield a consistent picture that accounts for the phenotypic heterogeneity observed over the course of drug tolerance development. The results reveal that, in certain plastic cancers, the population heterogeneity and evolution of cell phenotypes may be understood by accounting for the competing interactions of the epigenetic potential landscape and state-dependent cell proliferation. Accounting for such competition permits accurate, experimentally verifiable predictions that can potentially guide the design of effective treatment strategies.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Evolução Molecular , Melanoma , Fenótipo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Melanoma/genética , Melanoma/fisiopatologia , Modelos Biológicos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Serosurveys are useful for assessing population susceptibility to vaccine-preventable disease outbreaks. Although at-risk populations in remote areas could benefit from this type of information, they face several logistical barriers to implementation, such as lack of access to centralized laboratories, cold storage, and transport of samples. We describe a potential solution: a compact and portable, field-deployable, point-of-care system relying on digital microfluidics that can rapidly test a small volume of capillary blood for disease-specific antibodies. This system uses inexpensive, inkjet-printed digital microfluidic cartridges together with an integrated instrument to perform enzyme-linked immunosorbent assays (ELISAs). We performed a field validation of the system's analytical performance at Kakuma refugee camp, a remote setting in northwestern Kenya, where we tested children aged 9 to 59 months and caregivers for measles and rubella immunoglobulin G (IgG). The IgG assays were determined to have sensitivities of 86% [95% confidence interval (CI), 79 to 91% (measles)] and 81% [95% CI, 73 to 88% (rubella)] and specificities of 80% [95% CI, 49 to 94% (measles)] and 91% [95% CI, 76 to 97% (rubella)] (measles, n = 140; rubella, n = 135) compared with reference tests (measles IgG and rubella IgG ELISAs from Siemens Enzygnost) conducted in a centralized laboratory. These results demonstrate a potential role for this point-of-care system in global serological surveillance, particularly in remote areas with limited access to centralized laboratories.
Assuntos
Imunoensaio/métodos , Microfluídica/métodos , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Lactente , Masculino , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Despite advances in breast cancer prevention and treatment, variability in patient-response has revealed the need for a more "personalized" approach to medicine, in which treatments are tailored to each patient's biology. Motivated by this idea, we introduce a technique that allows for quantification of small-molecule analytes directly from core needle biopsy (CNB) tissue samples on a miniaturized platform. The new technique, powered by digital microfluidics, integrates tissue-liquid extraction and magnetic bead-based competitive immunoassay for quantification of estradiol in milligram-sized CNB samples. Each measurement (from start to finish) requires â¼40 minutes, a duration consistent with a visit to a doctor's office. The performance of the new technique was validated by the gold-standard analysis method (high performance liquid chromatography coupled to tandem mass spectrometry), and was applied to evaluate human patient samples before and after a course of treatment with aromatase inhibitor therapy. We propose that the new technique has great potential for eventual use for fast, automated, and quantitative analysis of biomarkers in tissue samples, towards a personalized medicine approach.
Assuntos
Inibidores da Aromatase/uso terapêutico , Biópsia com Agulha de Grande Calibre/instrumentação , Neoplasias da Mama , Monitoramento de Medicamentos/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Animais , Biópsia com Agulha de Grande Calibre/métodos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Desenho de Equipamento , Feminino , Humanos , RatosRESUMO
The diagnosis of infectious disease is typically carried out at the point-of-care (POC) using the lateral flow assay (LFA). While cost-effective and portable, LFAs often lack the clinical sensitivity and specificity required for accurate diagnoses. In response to this challenge, we introduce a new digital microfluidic (DMF) platform fabricated using a custom inkjet printing and roll-coating process that is scalable to mass production. The performance of the new devices is on par with that of traditional DMF devices fabricated in a cleanroom, with a materials cost for the new devices of only US $0.63 per device. To evaluate the usefulness of the new platform, we performed a 13-step rubella virus (RV) IgG immunoassay on the inkjet printed, roll-coated devices, which yielded a limit of detection of 0.02 IU mL-1, well below the diagnostic cut-off of 10 IU mL-1 for RV infection and immunity. We propose that this represents a breakthrough for DMF, lowering the costs to a level such that the new platforms will be an attractive alternative to LFAs for the diagnosis of infectious disease at the POC.
Assuntos
Custos e Análise de Custo , Tinta , Dispositivos Lab-On-A-Chip/economia , Sistemas Automatizados de Assistência Junto ao Leito , Impressão , Ensaio de Imunoadsorção Enzimática , Desenho de Equipamento , Imunoglobulina G/análise , Vírus da Rubéola/imunologia , Vírus da Rubéola/isolamento & purificaçãoRESUMO
Electrochemiluminescence (ECL) is a sensitive analytical technique with great promise for biological applications, especially when combined with microfluidics. Here, we report the first integration of ECL with digital microfluidics (DMF). ECL detectors were fabricated into the ITO-coated top plates of DMF devices, allowing for the generation of light from electrically excited luminophores in sample droplets. The new system was characterized by making electrochemical and ECL measurements of soluble mixtures of tris(phenanthroline)ruthenium(II) and tripropylamine (TPA) solutions. The system was then validated by application to an oligonucleotide hybridization assay, using magnetic particles bearing 21-mer, deoxyribose analogues of the complement to microRNA-143 (miRNA-143). The system detects single nucleotide mismatches with high specificity, and has a limit of detection of 1.5 femtomoles. The system is capable of detecting miRNA-143 in cancer cell lysates, allowing for the discrimination between the MCF-7 (less aggressive) and MDA-MB-231 (more aggressive) cell lines. We propose that DMF-ECL represents a valuable new tool in the microfluidics toolbox for a wide variety of applications.
Assuntos
Condutometria/instrumentação , Dispositivos Lab-On-A-Chip , Medições Luminescentes/instrumentação , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Experimentais/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Neoplasias Experimentais/diagnóstico , Neoplasias Experimentais/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Processamento de Sinais Assistido por Computador/instrumentaçãoRESUMO
Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF.
Assuntos
Técnicas de Cultura de Células/métodos , Técnicas Analíticas Microfluídicas/métodos , Adsorção , Técnicas de Cultura de Células/instrumentação , Separação Celular , Avaliação Pré-Clínica de Medicamentos , Eletroumectação , Humanos , Dispositivos Lab-On-A-Chip , Teste de Materiais , Poloxâmero , Proteínas/química , TensoativosRESUMO
We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the interrogation of protein phosphorylation on pulsing with stimulus for as little as 3 s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations-for example, a 10 s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population.
Assuntos
Fibroblastos/metabolismo , Imuno-Histoquímica/métodos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Humanos , Imuno-Histoquímica/instrumentação , Células MCF-7 , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Células NIH 3T3 , Fosforilação , Transdução de SinaisRESUMO
There is great interest in the development of integrated tools allowing for miniaturized sample processing, including solid phase extraction (SPE). We introduce a new format for microfluidic SPE relying on C18-functionalized magnetic beads that can be manipulated in droplets in a digital microfluidic platform. This format provides the opportunity to tune the amount (and potentially the type) of stationary phase on-the-fly, and allows the removal of beads after the extraction (to enable other operations in same device-space), maintaining device reconfigurability. Using the new method, we employed a design of experiments (DOE) operation to enable automated on-chip optimization of elution solvent composition for reversed phase SPE of a model system. Further, conditions were selected to enable on-chip fractionation of multiple analytes. Finally, the method was demonstrated to be useful for online cleanup of extracts from dried blood spot (DBS) samples. We anticipate this combination of features will prove useful for separating a wide range of analytes, from small molecules to peptides, from complex matrices.
Assuntos
Aminoquinolinas/análise , Angiotensina I/análise , Teste em Amostras de Sangue Seco , Nanopartículas de Magnetita/química , Técnicas Analíticas Microfluídicas , Solventes/química , Desenho de Equipamento , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Extração em Fase SólidaRESUMO
BACKGROUND: Whereas disease surveillance for infectious diseases such as rubella is important, it is critical to identify pregnant women at risk of passing rubella to their offspring, which can be fatal and can result in congenital rubella syndrome (CRS). The traditional centralized model for diagnosing rubella is cost-prohibitive in resource-limited settings, representing a major obstacle to the prevention of CRS. As a step toward decentralized diagnostic systems, we developed a proof-of-concept digital microfluidic (DMF) diagnostic platform that possesses the flexibility and performance of automated immunoassay platforms used in central facilities, but with a form factor the size of a shoebox. METHODS: DMF immunoassays were developed with integrated sample preparation for the detection of rubella virus (RV) IgG and IgM. The performance (sensitivity and specificity) of the assays was evaluated with serum and plasma samples from a commercial antirubella mixed-titer performance panel. RESULTS: The new platform performed the essential processing steps, including sample aliquoting for 4 parallel assays, sample dilution, and IgG blocking. Testing of performance panel samples yielded diagnostic sensitivity and specificity of 100% and 100% for both RV IgG and RV IgM. With 1.8 µL sample per assay, 4 parallel assays were performed in approximately 30 min with <10% mean CV. CONCLUSIONS: This proof of concept establishes DMF-powered immunoassays as being potentially useful for the diagnosis of infectious disease.
Assuntos
Anticorpos Antivirais/sangue , Imunoensaio/instrumentação , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Técnicas Analíticas Microfluídicas/instrumentação , Vírus da Rubéola/imunologia , Rubéola (Sarampo Alemão)/diagnóstico , Anticorpos Antivirais/imunologia , Desenho de Equipamento , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Gravidez , Rubéola (Sarampo Alemão)/sangue , Rubéola (Sarampo Alemão)/imunologia , Vírus da Rubéola/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
Many important biomarkers for disease diagnosis are present at low concentrations in human serum. These biomarkers are masked in proteomic analysis by highly abundant proteins such as human serum albumin (HSA) and immunoglobulins (IgGs) which account for up to 80% of the total protein content of serum. Traditional depletion methods using macro-scale LC-columns for highly abundant proteins involve slow separations which impart considerable dilution to the samples. Furthermore, most techniques lack the ability to process multiple samples simultaneously. We present a method of protein depletion using superparamagnetic beads coated in anti-HSA, Protein A, and Protein G, manipulated by digital microfluidics (DMF). The depletion process was capable of up to 95% protein depletion efficiency for IgG and HSA in 10 min for four samples simultaneously, which resulted in an approximately 4-fold increase in signal-to-noise ratio in MALDI-MS analysis for a low abundance protein, hemopexin. This rapid and automated method has the potential to greatly improve the process of biomarker identification.