RESUMO
Delivering macromolecules across biological barriers such as the blood-brain barrier limits their application in vivo. Previous work has demonstrated that Toxoplasma gondii, a parasite that naturally travels from the human gut to the central nervous system (CNS), can deliver proteins to host cells. Here we engineered T. gondii's endogenous secretion systems, the rhoptries and dense granules, to deliver multiple large (>100 kDa) therapeutic proteins into neurons via translational fusions to toxofilin and GRA16. We demonstrate delivery in cultured cells, brain organoids and in vivo, and probe protein activity using imaging, pull-down assays, scRNA-seq and fluorescent reporters. We demonstrate robust delivery after intraperitoneal administration in mice and characterize 3D distribution throughout the brain. As proof of concept, we demonstrate GRA16-mediated brain delivery of the MeCP2 protein, a putative therapeutic target for Rett syndrome. By characterizing the potential and current limitations of the system, we aim to guide future improvements that will be required for broader application.
Assuntos
Encéfalo , Neurônios , Proteínas de Protozoários , Toxoplasma , Toxoplasma/genética , Toxoplasma/metabolismo , Animais , Neurônios/metabolismo , Neurônios/parasitologia , Camundongos , Humanos , Encéfalo/metabolismo , Encéfalo/parasitologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Sistemas de Liberação de MedicamentosRESUMO
Human prion diseases are rare, transmissible and often rapidly progressive dementias. The most common type, sporadic Creutzfeldt-Jakob disease (sCJD), is highly variable in clinical duration and age at onset. Genetic determinants of late onset or slower progression might suggest new targets for research and therapeutics. We assembled and array genotyped sCJD cases diagnosed in life or at autopsy. Clinical duration (median:4, interquartile range (IQR):2.5-9 (months)) was available in 3,773 and age at onset (median:67, IQR:61-73 (years)) in 3,767 cases. Phenotypes were successfully transformed to approximate normal distributions allowing genome-wide analysis without statistical inflation. 53 SNPs achieved genome-wide significance for the clinical duration phenotype; all of which were located at chromosome 20 (top SNP rs1799990, pvalue = 3.45x10-36, beta = 0.34 for an additive model; rs1799990, pvalue = 9.92x10-67, beta = 0.84 for a heterozygous model). Fine mapping, conditional and expression analysis suggests that the well-known non-synonymous variant at codon 129 is the obvious outstanding genome-wide determinant of clinical duration. Pathway analysis and suggestive loci are described. No genome-wide significant SNP determinants of age at onset were found, but the HS6ST3 gene was significant (pvalue = 1.93 x 10-6) in a gene-based test. We found no evidence of genome-wide genetic correlation between case-control (disease risk factors) and case-only (determinants of phenotypes) studies. Relative to other common genetic variants, PRNP codon 129 is by far the outstanding modifier of CJD survival suggesting only modest or rare variant effects at other genetic loci.
Assuntos
Idade de Início , Síndrome de Creutzfeldt-Jakob , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Humanos , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/patologia , Idoso , Pessoa de Meia-Idade , Feminino , Masculino , Fenótipo , GenótipoRESUMO
Extracellularly released molecular inflammasome assemblies -ASC specks- cross-seed Aß amyloid in Alzheimer's disease. Here we show that ASC governs the extent of inflammation-induced amyloid A (AA) amyloidosis, a systemic disease caused by the aggregation and peripheral deposition of the acute-phase reactant serum amyloid A (SAA) in chronic inflammatory conditions. Using super-resolution microscopy, we found that ASC colocalized tightly with SAA in human AA amyloidosis. Recombinant ASC specks accelerated SAA fibril formation and mass spectrometry after limited proteolysis showed that ASC interacts with SAA via its pyrin domain (PYD). In a murine model of inflammatory AA amyloidosis, splenic amyloid load was conspicuously decreased in Pycard-/- mice which lack ASC. Treatment with anti-ASCPYD antibodies decreased amyloid loads in wild-type mice suffering from AA amyloidosis. The prevalence of natural anti-ASC IgG (-logEC50 ≥ 2) in 19,334 hospital patients was <0.01%, suggesting that anti-ASC antibody treatment modalities would not be confounded by natural autoimmunity. These findings expand the role played by ASC and IL-1 independent inflammasome employments to extraneural proteinopathies and suggest that anti-ASC immunotherapy may contribute to resolving such diseases.
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Commensal intestinal bacteria shape our microbiome and have decisive roles in preserving host metabolic and immune homeostasis. They conspicuously impact disease development and progression, including amyloid-beta (Aß) and alpha (α)-synuclein pathology in neurodegenerative diseases, conveying the importance of the brain-gut-microbiome axis in such conditions. However, little is known about the longitudinal microbiome landscape and its potential clinical implications in other protein misfolding disorders, such as prion disease. We investigated the microbiome architecture throughout prion disease course in mice. Fecal specimens were assessed by 16S ribosomal RNA sequencing. We report a temporal microbiome signature in prion disease and uncovered alterations in Lachnospiraceae, Ruminococcaceae, Desulfovibrionaceae, and Muribaculaceae family members in this disease. Moreover, we determined the enrichment of Bilophila, a microorganism connected to cognitive impairment, long before the clinical manifestation of disease symptoms. Based on temporal microbial abundances, several associated metabolic pathways and resulting metabolites, including short-chain fatty acids, were linked to the disease. We propose that neuroinflammatory processes relate to perturbations of the intestinal microbiome and metabolic state by an interorgan brain-gut crosstalk. Furthermore, we describe biomarkers possibly suitable for early disease diagnostics and anti-prion therapy monitoring. While our study is confined to prion disease, our discoveries might be of equivalent relevance in other proteinopathies and central nervous system pathologies.
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Iatrogenic transmission of prions, the infectious agents of fatal Creutzfeldt-Jakob disease, through inefficiently decontaminated medical instruments remains a critical issue. Harsh chemical treatments are effective, but not suited for routine reprocessing of reusable surgical instruments in medical cleaning and disinfection processes due to material incompatibilities. The identification of mild detergents with activity against prions is therefore of high interest but laborious due to the low throughput of traditional assays measuring prion infectivity. Here, we report the establishment of TESSA (sTainlESs steel-bead Seed Amplification assay), a modified real-time quaking induced cyclic amplification (RT-QuIC) assay that explores the propagation activity of prions with stainless steel beads. TESSA was applied for the screening of about 70 different commercially available and novel formulations and conditions for their prion inactivation efficacy. One hypochlorite-based formulation, two commercially available alkaline formulations and a manual alkaline pre-cleaner were found to be highly effective in inactivating prions under conditions simulating automated washer-disinfector cleaning processes. The efficacy of these formulations was confirmed in vivo in a murine prion infectivity bioassay, yielding a reduction of the prion titer for bead surface adsorbed prions below detectability. Our data suggest that TESSA represents an effective method for a rapid screening of prion-inactivating detergents, and that alkaline and oxidative formulations are promising in reducing the risk of potential iatrogenic prion transmission through insufficiently decontaminated instrument surfaces.
Assuntos
Príons , Aço Inoxidável , Instrumentos Cirúrgicos , Animais , Camundongos , Aço Inoxidável/química , Descontaminação/métodos , Síndrome de Creutzfeldt-Jakob/transmissão , Síndrome de Creutzfeldt-Jakob/prevenção & controle , Desinfecção/métodos , Detergentes/química , Detergentes/farmacologia , Humanos , Desinfetantes/farmacologia , OxirreduçãoRESUMO
Oligodendrocyte-lineage cells, including NG2 glia, undergo prominent changes in various neurodegenerative disorders. Here, we identify a neuroprotective role for NG2 glia against prion toxicity. NG2 glia were activated after prion infection in cerebellar organotypic cultured slices (COCS) and in brains of prion-inoculated mice. In both model systems, depletion of NG2 glia exacerbated prion-induced neurodegeneration and accelerated prion pathology. Loss of NG2 glia enhanced the biosynthesis of prostaglandin E2 (PGE2) by microglia, which augmented prion neurotoxicity through binding to the EP4 receptor. Pharmacological or genetic inhibition of PGE2 biosynthesis attenuated prion-induced neurodegeneration in COCS and mice, reduced the enhanced neurodegeneration in NG2-glia-depleted COCS after prion infection, and dampened the acceleration of prion disease in NG2-glia-depleted mice. These data unveil a non-cell-autonomous interaction between NG2 glia and microglia in prion disease and suggest that PGE2 signaling may represent an actionable target against prion diseases.
Assuntos
Dinoprostona , Microglia , Neuroglia , Neurônios , Doenças Priônicas , Transdução de Sinais , Animais , Microglia/metabolismo , Dinoprostona/metabolismo , Camundongos , Transdução de Sinais/fisiologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neuroglia/metabolismo , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Camundongos Endogâmicos C57BL , Antígenos/metabolismo , Proteoglicanas/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Príons/metabolismo , Príons/toxicidade , Cerebelo/metabolismo , Cerebelo/patologiaRESUMO
AIMS OF THE STUDY: Systemic amyloidoses are rare protein-folding diseases with heterogeneous, often nonspecific clinical presentations. To better understand systemic amyloidoses and to apply state-of-the-art diagnostic pathways and treatment, the interdisciplinary Amyloidosis Network was founded in 2013 at University Hospital Zurich. In this respect, a registry was implemented to study the characteristics and life expectancy of patients with amyloidosis within the area covered by the network. Patient data were collected retrospectively for the period 2005-2014 and prospectively from 2015 onwards. METHODS: Patients aged 18 years or older diagnosed with any subtype of systemic amyloidosis were eligible for inclusion if they were treated in one of the four referring centres (Zurich, Chur, St Gallen, Bellinzona). Baseline data were captured at the time of diagnosis. Follow-up data were assessed half-yearly for the first two years, then annually. RESULTS: Between January 2005 and March 2020, 247 patients were screened, and 155 patients with confirmed systemic amyloidosis were included in the present analysis. The most common amyloidosis type was light-chain (49.7%, n = 77), followed by transthyretin amyloidosis (40%, n = 62) and amyloid A amyloidosis (5.2%, n = 8). Most patients (61.9%, n = 96) presented with multiorgan involvement. Nevertheless, single organ involvement was seen in all types of amyloidosis, most commonly in amyloid A amyloidosis (75%, n = 6). The median observation time of the surviving patients was calculated by the reverse Kaplan-Meier method and was 3.29 years (95% confidence interval [CI] 2.33-4.87); it was 4.87 years (95% CI 3.14-7.22) in light-chain amyloidosis patients and 1.85 years (95% CI 1.48-3.66) in transthyretin amyloidosis patients, respectively. The 1-, 3- and 5-year survival rates were 87.0% (95% CI 79.4-95.3%), 68.5% (95% CI 57.4-81.7%) and 66.0% (95% CI 54.6-79.9%) respectively for light-chain amyloidosis patients and 91.2% (95% CI 83.2-99.8%), 77.0% (95% CI 63.4-93.7%) and 50.6% (95% CI 31.8-80.3%) respectively for transthyretin amyloidosis patients. There was no significant difference between the two groups (p = 0.81). CONCLUSION: During registry set-up, a more comprehensive work-up of our patients suffering mainly from light-chain amyloidosis and transthyretin amyloidosis was implemented. Survival rates were remarkably high and similar between light-chain amyloidosis and transthyretin amyloidosis, a finding which was noted in similar historic registries of international centres. However, further studies are needed to depict morbidity and mortality as the amyloidosis landscape is changing rapidly.
Assuntos
Neuropatias Amiloides Familiares , Amiloidose , Humanos , Neuropatias Amiloides Familiares/diagnóstico , Neuropatias Amiloides Familiares/metabolismo , Neuropatias Amiloides Familiares/terapia , Sistema de Registros , Estudos Retrospectivos , Proteína Amiloide A Sérica , Suíça/epidemiologia , AdultoRESUMO
In 2015, we launched the mesoSPIM initiative, an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of such microscopes. Here, we introduce the next-generation mesoSPIM ("Benchtop") with a significantly increased field of view, improved resolution, higher throughput, more affordable cost, and simpler assembly compared to the original version. We develop an optical method for testing detection objectives that enables us to select objectives optimal for light-sheet imaging with large-sensor cameras. The improved mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, magnification up to 20×, and supports sample sizes ranging from sub-mm up to several centimeters while being compatible with multiple clearing techniques. The microscope serves a broad range of applications in neuroscience, developmental biology, pathology, and even physics.
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Microscopia , Neurociências , Microscopia/métodosRESUMO
Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1, which involve human-specific mechanisms2-5 that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors6. Single-cell transcriptomics and comparison to independent neural stem cells7 showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids8. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C-Reativa , Proteínas de Ligação a DNA , Degeneração Lobar Frontotemporal , Rede Nervosa , Proteínas do Tecido Nervoso , Neurônios , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteína C-Reativa/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/patologia , Rede Nervosa/metabolismo , Rede Nervosa/patologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neurônios/citologia , Neurônios/metabolismo , Reprodutibilidade dos TestesRESUMO
Descriptive data are rapidly expanding in biomedical research. Instead, functional validation methods with sufficient complexity remain underdeveloped. Transcriptional reporters allow experimental characterization and manipulation of developmental and disease cell states, but their design lacks flexibility. Here, we report logical design of synthetic cis-regulatory DNA (LSD), a computational framework leveraging phenotypic biomarkers and trans-regulatory networks as input to design reporters marking the activity of selected cellular states and pathways. LSD uses bulk or single-cell biomarkers and a reference genome or custom cis-regulatory DNA datasets with user-defined boundary regions. By benchmarking validated reporters, we integrate LSD with a computational ranking of phenotypic specificity of putative cis-regulatory DNA. Experimentally, LSD-designed reporters targeting a wide range of cell states are functional without minimal promoters. Applied to broadly expressed genes from human and mouse tissues, LSD generates functional housekeeper-like sLCRs compatible with size constraints of AAV vectors for gene therapy applications. A mesenchymal glioblastoma reporter designed by LSD outperforms previously validated ones and canonical cell surface markers. In genome-scale CRISPRa screens, LSD facilitates the discovery of known and novel bona fide cell-state drivers. Thus, LSD captures core principles of cis-regulation and is broadly applicable to studying complex cell states and mechanisms of transcriptional regulation.
Assuntos
DNA , Regulação da Expressão Gênica , Animais , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , Expressão Gênica , BiomarcadoresRESUMO
Recanalization is the mainstay of ischemic stroke treatment. However, even with timely clot removal, many stroke patients recover poorly. Leptomeningeal collaterals (LMCs) are pial anastomotic vessels with yet-unknown functions. We applied laser speckle imaging, ultrafast ultrasound, and two-photon microscopy in a thrombin-based mouse model of stroke and fibrinolytic treatment to show that LMCs maintain cerebral autoregulation and allow for gradual reperfusion, resulting in small infarcts. In mice with poor LMCs, distal arterial segments collapse, and deleterious hyperemia causes hemorrhage and mortality after recanalization. In silico analyses confirm the relevance of LMCs for preserving perfusion in the ischemic region. Accordingly, in stroke patients with poor collaterals undergoing thrombectomy, rapid reperfusion resulted in hemorrhagic transformation and unfavorable recovery. Thus, we identify LMCs as key components regulating reperfusion and preventing futile recanalization after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for beneficial reperfusion after stroke.
Assuntos
Circulação Colateral , AVC Isquêmico , Meninges , Reperfusão , Animais , AVC Isquêmico/fisiopatologia , AVC Isquêmico/terapia , Camundongos , Circulação Colateral/fisiologia , Humanos , Reperfusão/métodos , Meninges/irrigação sanguínea , Masculino , Circulação Cerebrovascular/fisiologia , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Encéfalo/irrigação sanguínea , Trombectomia/métodosRESUMO
Long Covid is a debilitating condition of unknown etiology. We performed multimodal proteomics analyses of blood serum from COVID-19 patients followed up to 12 months after confirmed severe acute respiratory syndrome coronavirus 2 infection. Analysis of >6500 proteins in 268 longitudinal samples revealed dysregulated activation of the complement system, an innate immune protection and homeostasis mechanism, in individuals experiencing Long Covid. Thus, active Long Covid was characterized by terminal complement system dysregulation and ongoing activation of the alternative and classical complement pathways, the latter associated with increased antibody titers against several herpesviruses possibly stimulating this pathway. Moreover, markers of hemolysis, tissue injury, platelet activation, and monocyte-platelet aggregates were increased in Long Covid. Machine learning confirmed complement and thromboinflammatory proteins as top biomarkers, warranting diagnostic and therapeutic interrogation of these systems.
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Ativação do Complemento , Proteínas do Sistema Complemento , Síndrome de COVID-19 Pós-Aguda , Proteoma , Tromboinflamação , Humanos , Proteínas do Sistema Complemento/análise , Proteínas do Sistema Complemento/metabolismo , Síndrome de COVID-19 Pós-Aguda/sangue , Síndrome de COVID-19 Pós-Aguda/complicações , Síndrome de COVID-19 Pós-Aguda/imunologia , Tromboinflamação/sangue , Tromboinflamação/imunologia , Biomarcadores/sangue , Proteômica , Masculino , Feminino , Adulto Jovem , Adulto , Pessoa de Meia-Idade , IdosoRESUMO
My first encounter with prion diseases dates to 1986. As a clinical resident in neuropathology, I was tasked with performing autopsies of patients who died of mysterious brain diseases. In his early 60s, my patient had developed a form of dementia that progressed at a terrifyingly rapid pace and eventually led to his death. I sampled the patient's brain and processed it for histological examination. The microscope revealed an eerie landscape of destruction. All that was left in the patient's cortex were astrocytes and microglia, and the few remaining neurons showed extensive vacuolation of their bodies and processes. Such blazing destruction of the brain was indicative of just one diagnosis: Creutzfeldt-Jakob disease, a spongiform encephalopathy caused by enigmatic infectious agents called prions.
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Doenças Priônicas , Príons , Humanos , Córtex Cerebral/patologia , Síndrome de Creutzfeldt-Jakob/história , Doenças Priônicas/história , Príons/históriaRESUMO
Prion diseases are fatal neurodegenerative disorders characterized by the conversion of the cellular prion protein (PrPC) into a misfolded prion form, which is believed to disrupt the cellular membranes. However, the exact mechanisms underlying prion toxicity, including the formation of membrane pores, are not fully understood. The prion protein consists of two domains: a globular domain (GD) and a flexible N-terminus (FT) domain. Although a proximal polybasic amino acid (FT(23-31) sequence of FT is a prerequisite for cellular membrane permeabilization, other functional domain regions may modulate its effects. Through single-channel electrical recordings and cryo-electron microscopy (cryo-EM), we discovered that the FT(23-50) fragment forms pore-shaped oligomers and plays a dominant role in membrane permeabilization within the full-length mouse prion protein (mPrP(23-230)). In contrast, the FT(51-110) domain or the C-terminal domain downregulate the channel activity of FT(23-50) and mPrP(23-230). The addition of prion mimetic antibody, POM1 significantly amplifies mPrP(23-230) membrane permeabilization, whereas POM1_Y104A, a mutant that binds to PrP but cannot elicit toxicity, has a negligible effect on membrane permeabilization. Additionally, the anti-N-terminal antibody POM2 or Cu2+ binds to the FT domain, subsequently enhancing the FT(23-110) channel activity. Importantly, our setup provides a novel approach without an external fused protein to examine the channel activity of truncated PrP in the lipid membranes. We therefore propose that the primary N-terminal residues are essential for membrane permeabilization, while other functional segments of PrP play a vital role in modulating the pathological effects of PrP-mediated neurotoxicity.
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Proteínas PrPC , Doenças Priônicas , Príons , Camundongos , Animais , Príons/metabolismo , Proteínas Priônicas/genética , Microscopia Crioeletrônica , Membrana Celular/metabolismo , Anticorpos , Proteínas PrPC/químicaRESUMO
Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy.
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Telescópios , Animais , Imersão , Microscopia/métodos , Peixe-ZebraRESUMO
There are no cures for the most common neurodegenerative diseases. None of the currently approved treatments cure or halt these conditions; rather, they address symptoms or slow disease progression. A focus on protein deposits in the brain-a hallmark of Alzheimer's disease (AD) and Parkinson's disease (PD)-has led to the development of immunotherapy drugs. Other promising avenues of investigation include the roles of neuroinflammation in neurodegeneration. However, the clinical impact of these approaches is still uncertain. What about exploiting our knowledge of the human genome and the ability to modify it with surgically precise tools? Can functional genomics approaches in neurodegenerative disease research provide the breakthroughs we need?
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Doença de Alzheimer , Doença de Parkinson , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Encéfalo , Genoma Humano , Genômica , Doença de Parkinson/genética , Doença de Parkinson/terapiaRESUMO
Conventional methods of measuring affinity are limited by artificial immobilization, large sample volumes, and homogeneous solutions. This protocol describes microfluidic antibody affinity profiling on complex human samples in solution to obtain a fingerprint reflecting both affinity and active concentration of the target protein. To illustrate the protocol, we analyze the antibody response in SARS-CoV-2 omicron-naïve samples against different SARS-CoV-2 variants of concern. However, the protocol and the technology are amenable to a broad spectrum of biomedical questions. For complete details on the use and execution of this protocol, please refer to Emmenegger et al. (2022),1 Schneider et al. (2022),2 and Fiedler et al. (2022).3.