Bead-based Immunoassay Research Articles

Hybrid Antibody-Aptamer Assay for Detection of Tetrodotoxin in Pufferfish.

The marine toxin tetrodotoxin (TTX) poses a great risk to public health safety due to its severe paralytic effects after ingestion. Seafood poisoning caused by the consumption of contaminated marine species like pufferfish due to its expansion to nonendemic areas has increased the need for fast and reliable detection of the toxin to effectively implement prevention strategies. Liquid chromatography-mass spectrometry is considered the most accurate method, although competitive immunoassays have also been reported. In this work, we sought to develop an aptamer-based assay for the rapid, sensitive, and cost-effective detection of TTX in pufferfish. Using capture-SELEX combined with next-generation sequencing, aptamers were identified, and their binding properties were evaluated. Finally, a highly sensitive and user-friendly hybrid antibody–aptamer sandwich assay was developed with superior performance compared to several assays reported in the literature and commercial immunoassay kits. The assay was successfully applied to the quantification of TTX in pufferfish extracts, and the results obtained correlated very well with a competitive magnetic bead-based immunoassay performed in parallel for comparison. This is one of the very few works reported in the literature of such hybrid assays for small-molecule analytes whose compatibility with field samples is also demonstrated.

Purification of native histidine-rich protein 2 (nHRP2) from Plasmodium falciparum culture supernatant, infected RBCs, and parasite lysate

BackgroundDespite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field.MethodsThis report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay.ResultsPurified nHRP2 was identified by SDS-PAGE and western blot as a − 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL.ConclusionsPurified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.Graphical

Comparison study of bead-based and line-blot multiplex ANA immunoassays in the diagnosis of systemic autoimmune rheumatic diseases

Detection of antinuclear antibodies (ANA) by immunofluorescence assay (IFA) is increasingly substituted by multiplex bead-based immunoassay (MBA) and line-blot immunoassay (LIA). This study is to compare the diagnostic performance of MBA and LIA ANA assays on clinically characterized patient samples. A total of 728 serum samples from 385 patients with systemic autoimmune rheumatic diseases (SARD), 204 patients with non-SARD diseases, and 139 apparently healthy subjects were tested with the BioPlex 2200 ANA Screen and EuroLine ANA Profile 3 as the representative MBA and LIA technologies and HEp-2 ANA IFA. Clinical data were collected independent of laboratory analysis and later related to the ANA test results. The clinical diagnostic performances were analyzed using Analyse-it software. The MBA demonstrated higher area under curve (AUC) compared to LIA (0.814 vs 0.761, p = 0.002) and HEp-2 IFA (0.814 vs 0.771, p = 0.008). The MBA and LIA ANA methods showed higher specificity (83.8% and 77.0% vs 67.6%, p < 0.001 and p = 0.005) but lower sensitivity (79.0% and 75.3% vs 86.5%, p < 0.001) compared to HEp-2 IFA. The MBA and LIA ANA revealed substantial to excellent agreements on specific antinuclear antibodies except anti-dsDNA, with the total agreement from 92.3 to 99.9% and Cohen's kappa from 0.71 to 0.98. The MBA demonstrated significantly higher sensitivity (58.1% vs 19.8%, p < 0.001) and comparable specificity (95.9% vs 97.5%, p = 0.221) on anti-dsDNA assay for the diagnosis of SLE compared to LIA. The MBA and LIA ANA assays have higher specificity but lower sensitivity compared to HEp-2 IFA. There are good agreements between MBA and LIA ANA for the specific antinuclear antibodies except for anti-dsDNA. The MBA ANA demonstrated better assay performance compared to LIA as the MBA possesses higher sensitivity and specificity in the diagnosis of SARD. Key Points • The multiplex bead-based immunoassay (MBA) ANA outperformed line-blot immunoassay (LIA) and traditional HEp-2 IFA. • There are good agreements between the MBA BioPlex 2200 ANA Screen and LIA EuroLine ANA Profile 3 for the most of specific antinuclear antibodies except anti-dsDNA. • Additional anti-dsDNA testing is suggested when EuroLine ANA Profile 3 is used for the aid of SLE diagnosis and management. • The positive predictive value of both multiplex ANA assays can be substantially increased without significantlyaffecting negative predictive value by using at least two specific antinuclear antibodies for reporting a positive ANAresult.

Cytokine response in cerebrospinal fluid of meningitis patients and outcome associated with pneumococcal serotype

Streptococcus pneumoniae causes life-threatening meningitis. Its capsular polysaccharide determines the serotype and influences disease severity but the mechanism is largely unknown. Due to evidence of elevated cytokines levels in the meningeal inflammatory response, we measured 41 cytokines/chemokines and growth factors in cerebrospinal fluid (CSF) samples from 57 South African meningitis patients (collected in the period 2018–2019), with confirmed S. pneumoniae serotypes, using a multiplexed bead-based immunoassay. Based on multivariable Bayesian regression, using serotype 10A as a reference and after adjusting for HIV and age, we found IL-6 concentrations significantly lower in patients infected with serotypes 6D (undetectable) and 23A (1601 pg/ml), IL-8 concentrations significantly higher in those infected with 22A (40,459 pg/ml), 7F (32,400 pg/ml) and 15B/C (6845 pg/ml), and TNFα concentration significantly higher in those infected with serotype 18A (33,097 pg/ml). Although a relatively small number of clinical samples were available for this study and 28% of samples could not be assigned to a definitive serotype, our data suggests 15B/C worthy of monitoring during surveillance as it is associated with in-hospital case fatality and not included in the 13-valent polysaccharide conjugate vaccine, PCV13. Our data provides average CSF concentrations of a range of cytokines and growth factors for 18 different serotypes (14, 19F, 3, 6A, 7F, 19A, 8, 9N, 10A, 12F, 15B/C, 22F, 16F, 23A, 31, 18A, 6D, 22A) to serve as a basis for future studies investigating host–pathogen interaction during pneumococcal meningitis. We note that differences in induction of IL-8 between serotypes may be particularly worthy of future study.

Dual effects of interleukin-10 on natural killer cells and monocytes and the implications for adult-onset Still's disease.

We compared the serum levels of multiple cytokines in patients with adult-onset Still's disease (AOSD) and healthy controls to assess the effects of humoral factors on natural killer (NK) cells and monocytes. We quantified the serum levels of 10 cytokines in the patients using bead-based multiplex immunoassays, along with interleukin (IL-)18 using ELISA. We then sorted NK cells and monocytes from the peripheral blood mononuclear cells (PBMCs) of healthy volunteers, cultured them in the presence or absence of cytokines that were detected in some or all of the serum samples from the AOSD patients and their combinations in vitro, and analysed the culture supernatant. IL-6 and IL-18 were the main cytokines increased in the serum of AOSD patients. When NK cells were cultured with the cytokines and IL-10, the combination of IL-10 and IL-18 substantially induced interferon (IFN-)γ. IL-6 had little effect on NK cells, probably because they barely expressed the IL-6 receptor and glycoprotein 130 (gp130). IFN-γ induced monocytes to produce IL-1β, IL-6 and tumour necrosis factor (TNF-)α whereas IL-10 inhibited the induction of these proinflammatory cytokines. IL-10 evidently has dual effects on NK cells (stimulation) and on monocytes (inhibition). Better understanding the roles of the cytokine network would shed light on the pathogenesis of AOSD.

Changes in systemic GDF15 across the adult lifespan and their impact on maximal muscle power: the Copenhagen Sarcopenia Study

BackgroundAlthough growth differentiation factor 15 (GDF15) is known to increase with disease and is associated with low physical performance, the role of GDF15 in normal ageing is still not fully understood. Specifically, the influence of circulating GDF15 on impairments in maximal muscle power (a major contributor to functional limitations) and the underlying components has not been investigated.MethodsData from 1305 healthy women and men aged 20 to 93 years from The Copenhagen Sarcopenia Study were analysed. Circulating levels of GDF15 and markers of inflammation (tumor necrosis factor‐alpha, interleukin‐6, and high‐sensitivity C‐reactive protein) were measured by ELISA (R&D Systems) and multiplex bead‐based immunoassays (Bio‐Rad). Relative (normalized to body mass), allometric (normalized to height squared), and specific (normalized to leg muscle mass) muscle power were assessed by the Nottingham power rig [leg extension power (LEP)] and the 30 s sit‐to‐stand (STS) muscle power test. Total body fat, visceral fat, and leg lean mass were assessed by dual energy X‐ray absorptiometry. Leg skeletal muscle index was measured as leg lean mass normalized to body height squared.ResultsSystemic levels of GDF15 increased progressively as a function of age in women (1.1 ± 0.4 pg·mL−1·year−1) and men (3.3 ± 0.6 pg·mL−1·year−1) (both P < 0.05). Notably, GDF15 increased at a faster rate from the age of 65 years in women (11.5 ± 1.2 pg·mL−1·year−1, P < 0.05) and 70 years in men (19.3 ± 2.3 pg·mL−1·year−1, P < 0.05), resulting in higher GDF15 levels in men compared with women above the age of 65 years (P < 0.05). Independently of age and circulatory markers of inflammation, GDF15 was negatively correlated to relative STS power (P < 0.05) but not LEP, in both women and men. These findings were mainly explained by negative associations of GDF15 with specific STS power in women and men (both P < 0.05).ConclusionsA J‐shaped relationship between age and systemic GDF15 was observed, with men at older age showing steeper increases and elevated GDF15 levels compared with women. Importantly, circulating GDF15 was independently and negatively associated with relative STS power, supporting the potential role of GDF15 as a sensitive biomarker of frailty in older people.

ECTRIMS 2021 – Late Breaking News ePoster

Objective: To compare humoral and T-cell responses to COVID- 19 vaccines in 400 MS patients who were on Ocrelizumab ('OCR') v. other disease-modifying therapies ('non-OCR') at the time of vaccination. Introduction: Peripheral B-cell depletion with anti-CD20 therapies attenuates humoral responses to vaccines. Whether immune responses to COVID-19 vaccines differ between B-cell depleted and non-B cell depleted MS patients is not known. Methods: Consecutive MS patients from NYU MS Care Center were invited to participate if they completed COVID-19 vaccination ≥6 weeks previously. Immune testing included anti-spike RBD antibody (Elecsys Anti-SARS-CoV-2) (Roche Diagnostics);multiplex bead-based immunoassays of antibody-responses to SARS-COV-2 spike proteins;T-cell responses to SARS-CoV-2 Spike protein using IFNγ enzyme-linked immune-absorbent spot (Invitrogen) and TruCulture (Myriad RBM) assays;high dimensional immunophenotyping;and live virus immunofluorescencebased microneutralization assay. Results: As of 7/15/2021, 105 MS subjects were enrolled (mean age: 40.5 years;76% female;41% non-white;38% on OCR;12% with prior COVID-19 infection). 95% were fully vaccinated with mRNA vaccines (Pfizer/Moderna);5% - with adenovirus-based vaccine (JohnsonJp<0.0001). Positive response by multiplex assay (threshold of 'positive' defined as 2 SD below the mean for the non-OCR) were detected in 10/27 (37%) OCR and 29/31 (94%) non-OCR (p<0.00001). T-cell activation based on induced IFNγ secretion (TruCulture) was detected in 20/25 (80%) OCR and 16/19 (84%) non-OCR patients (p=0.71). Conclusions: Preliminary results suggest robust T-cell immune response to SARS-CoV2 vaccines in approximately 80% of both OCR and non-OCR MS patients. Antibody responses were markedly attenuated in OCR compared to non-OCR group. Updated results will be presented.

082 Bath-PUVA therapy ameliorates pathogenetic chemokines for psoriasis

Psoriasis is a T-cell mediated autoimmune disease characterized by increased keratinocyte proliferation. In psoriasis, chemokines are secreted by a wide variety of cells (e.g. keratinocytes, dendritic cells, monocytes), which migrate immune cells (Th17 cells, Th1 cells, neutrophils, etc.) involved in the pathogenesis. Bath-psoralen plus ultraviolet A (PUVA) treatment is an established phototherapy for psoriasis, but its effects on chemokine levels remain unknown. We measured the levels of 22 serum chemokines by using a flow cytometry bead-based immunoassay in 20 psoriasis patients first treated with bath-PUVA therapy between 2007 and 2011 in our hospital.

Bio-P-13 - Boost of innate immunity cytokines as biomarkers of response to extracorporeal photopheresis in leukemic cutaneous T-cell lymphoma patients

Extracorporeal photopheresis (ECP) emerged as a systemic first-line therapy in leukemic cutaneous T-cell lymphoma (L-CTCL) and is now being implemented in a variety of T-cell-mediated diseases. Although ECP has been used for nearly 30 years, the mechanisms of its efficacy are not sufficiently understood and biomarkers for response are lacking. We aim to investigate the immunomodulatory effects of ECP on cytokine secretion patterns in patients with L-CTCL. A total of 22 L-CTCL patients and 15 healthy donors (HDs) were retrospectively enrolled in this study. Concentrations of 16 cytokines were simultaneously quantified by using multiplex bead-based immunoassays. First, the result revealed distinct cytokine patterns between L-CTCLs and HDs. We observed significant loss of TNFa and IL-8, and significant increase of IL-9 and IL-13 in serums of L-CTCL patients compare to HDs. As a next step, L-CTCL patients who received ECP were classified to responder and non-responder to treatment according to their quantitative reduction of malignant burden in the blood. We evaluated cytokine levels in culture supernatants from patient peripheral blood mononuclear cells (PBMCs) at baseline and over 27 weeks after ECP. Strikingly, PBMCs purified from ECP responders released statistically higher concentration of innate immunity cytokines such as IL-1a, IL-1b, GM-CSF and TNFα in comparison with ECP non-responders. In parallel, clinical observations documented complete clearance of erythema, reduction of malignant clonal T cells in the blood, and a potent boost of relevant innate immunity cytokines in individual L-CTCL patients. To conclude, our results demonstrated that ECP stimulates innate immune network, and this implied how ECP facilitates redirection of tumor-biased immunosuppressive microenvironment toward active anti-tumor responses. The alteration of TNFα, IL-1a, IL-1b and GM-CSF can possibly be used as biomarkers of response to ECP in leukemic CTCL patients.

Plasmodium falciparum is evolving to escape malaria rapid diagnostic tests in Ethiopia

In Africa, most rapid diagnostic tests (RDTs) for falciparum malaria recognize histidine-rich protein 2 antigen. Plasmodium falciparum parasites lacking histidine-rich protein 2 (pfhrp2) and 3 (pfhrp3) genes escape detection by these RDTs, but it is not known whether these deletions confer sufficient selective advantage to drive rapid population expansion. By studying blood samples from a cohort of 12,572 participants enroled in a prospective, cross-sectional survey along Ethiopia’s borders with Eritrea, Sudan and South Sudan using RDTs, PCR, an ultrasensitive bead-based immunoassay for antigen detection and next-generation sequencing, we estimate that histidine-rich protein 2-based RDTs would miss 9.7% (95% confidence interval 8.5–11.1) of P. falciparum malaria cases owing to pfhrp2 deletion. We applied a molecular inversion probe-targeted deep sequencing approach to identify distinct subtelomeric deletion patterns and well-established pfhrp3 deletions and to uncover recent expansion of a singular pfhrp2 deletion in all regions sampled. We propose a model in which pfhrp3 deletions have arisen independently multiple times, followed by strong positive selection for pfhrp2 deletion owing to RDT-based test-and-treatment. Existing diagnostic strategies need to be urgently reconsidered in Ethiopia, and improved surveillance for pfhrp2 deletion is needed throughout the Horn of Africa.

Adaptation of ELISA detection of Plasmodium falciparum and Plasmodium vivax circumsporozoite proteins in mosquitoes to a multiplex bead-based immunoassay

BackgroundPlasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of Plasmodium falciparum, Plasmodium vivax VK210 (P. vivax210) or P. vivax VK247 (P. vivax247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites.MethodsRecombinant positive controls for P. falciparum, P. vivax210 and P. vivax247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cut-off measures were applied to demonstrate how cut-off criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins.ResultsFollowing optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for P. falciparum, P. vivax210 and P. vivax247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for P. falciparum, 425 for P. vivax210 and 1650 for P. vivax247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4 and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decrease in the Madagascar sporozoite rate to 2.8–4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cut-off value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cut-off value is determined.ConclusionsThe csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to P. falciparum, P. vivax210 and P. vivax247 can be added following optimization.

Fluorescent Multiplex Immunohistochemistry Coupled With Other State-Of-The-Art Techniques to Systematically Characterize the Tumor Immune Microenvironment.

Our expanding knowledge of the interactions between tumor cells and their microenvironment has helped to revolutionize cancer treatments, including the more recent development of immunotherapies. Immune cells are an important component of the tumor microenvironment that influence progression and treatment responses, particularly to the new immunotherapies. Technological advances that help to decipher the complexity and diversity of the tumor immune microenvironment (TIME) are increasingly used in translational research and biomarker studies. Current techniques that facilitate TIME evaluation include flow cytometry, multiplex bead-based immunoassays, chromogenic immunohistochemistry (IHC), fluorescent multiplex IHC, immunofluorescence, and spatial transcriptomics. This article offers an overview of our representative data, discusses the application of each approach to studies of the TIME, including their advantages and challenges, and reviews the potential clinical applications. Flow cytometry and chromogenic and fluorescent multiplex IHC were used to immune profile a HER2+ breast cancer, illustrating some points. Spatial transcriptomic analysis of a luminal B breast tumor demonstrated that important additional insight can be gained from this new technique. Finally, the development of a multiplex panel to identify proliferating B cells, Tfh, and Tfr cells on the same tissue section demonstrates their co-localization in tertiary lymphoid structures.

Antibody Responses to SARS-CoV-2 After Infection or Vaccination in Children and Young Adults With Inflammatory Bowel Disease.

BackgroundCharacterization of neutralization antibodies to SARS-CoV-2 infection or vaccination in children and young adults with inflammatory bowel disease (IBD) receiving biologic therapies is crucial.MethodsWe performed a prospective longitudinal cohort study evaluating SARS-CoV-2 spike protein receptor binding domain (S-RBD) IgG positivity along with consistent clinical symptoms in patients with IBD receiving infliximab or vedolizumab. Serum was also obtained following immunization with approved vaccines. The IgG antibody to the spike protein binding domain of SARS-CoV-2 was assayed with a fluorescent bead-based immunoassay that takes advantage of the high dynamic range of fluorescent molecules using flow cytometry. A sensitive and high-throughput neutralization assay that incorporates SARS-CoV-2 spike protein onto a lentivirus and measures pseudoviral entry into ACE2-angiotensin converting enzyme 2 (ACE2) expressing human embryonic kidney 293 (HEK-293) cells was used.ResultsThere were 436 patients enrolled (mean age, 17 years, range 2–26 years; 58% male; 71% Crohn’s disease, 29% ulcerative colitis, IBD-unspecified). Forty-four (10%) of enrolled subjects had SARS-CoV-2 S-RBD IgG antibodies. Compared to non-IBD adults (ambulatory) and hospitalized pediatric patients with PCR documented SARS-CoV-2 infection, S-RBD IgG antibody levels were significantly lower in the IBD cohort and by 6 months post infection most patients lacked neutralizing antibody. Following vaccination (n = 33), patients had a 15-fold higher S-RBD antibody response in comparison with natural infection, and all developed neutralizing antibodies to both wild type and variant SARS-CoV-2.ConclusionsThe lower and less durable SARS-CoV-2 S-RBD IgG response to natural infection in IBD patients receiving biologics puts them at risk of reinfection. The robust response to immunization is likely protective.

Digital immunoassay for biomarker concentration quantification using solid-state nanopores

ABSTRACTSingle-molecule counting is the most accurate and precise method for determining the concentration of a biomarker in solution and is leading to the emergence of digital diagnostic platforms enabling precision medicine. In principle, solid-state nanopores—fully electronic sensors with single-molecule sensitivity—are well suited to the task. Here we present a digital immunoassay scheme capable of reliably quantifying the concentration of a target protein in complex biofluids that overcomes specificity, sensitivity, and consistency challenges associated with the use of solid-state nanopores for protein sensing. This is achieved by employing easily-identifiable DNA nanostructures as proxies for the presence (“1”) or absence (“0”) of the target protein captured via a magnetic bead-based sandwich immunoassay. As a proof-of-concept, we demonstrate quantification of the concentration of thyroid-stimulating hormone from human serum samples down to the high femtomolar range. Further optimization to the method will push sensitivity and dynamic range, allowing for development of precision diagnostic tools compatible with point-of-care format.

A Variable Height Microfluidic Device for Multiplexed Immunoassay Analysis of Traumatic Brain Injury Biomarkers.

Traumatic brain injury (TBI) is a leading cause of global morbidity and mortality, partially due to the lack of sensitive diagnostic methods and efficacious therapies. Panels of protein biomarkers have been proposed as a way of diagnosing and monitoring TBI. To measure multiple TBI biomarkers simultaneously, we present a variable height microfluidic device consisting of a single channel that varies in height between the inlet and outlet and can passively multiplex bead-based immunoassays by trapping assay beads at the point where their diameter matches the channel height. We developed bead-based quantum dot-linked immunosorbent assays (QLISAs) for interleukin-6 (IL-6), glial fibrillary acidic protein (GFAP), and interleukin-8 (IL-8) using DynabeadsTM M-450, M-270, and MyOneTM, respectively. The IL-6 and GFAP QLISAs were successfully multiplexed using a variable height channel that ranged in height from ~7.6 µm at the inlet to ~2.1 µm at the outlet. The IL-6, GFAP, and IL-8 QLISAs were also multiplexed using a channel that ranged in height from ~6.3 µm at the inlet to ~0.9 µm at the outlet. Our system can keep pace with TBI biomarker discovery and validation, as additional protein biomarkers can be multiplexed simply by adding in antibody-conjugated beads of different diameters.

Expression of IL-20 Receptor Subunit β Is Linked to EAE Neuropathology and CNS Neuroinflammation.

Considerable clinical evidence supports that increased blood–brain barrier (BBB) permeability is linked to immune extravasation of CNS parenchyma during neuroinflammation. Although BBB permeability and immune extravasation are known to be provoked by vascular endothelial growth factor-A (i.e., VEGF-A) and C-X-C motif chemokine ligand 12 (CXCL12), respectively, the mechanisms that link both processes are still elusive. The interleukin-20 (i.e., IL-20) cytokine signaling pathway was previously implicated in VEGF-mediated angiogenesis and is known to induce cellular response by way of signaling through IL-20 receptor subunit β (i.e., IL-20RB). Dysregulated IL-20 signaling is implicated in many inflammatory pathologies, but it’s contribution to neuroinflammation has yet to be reported. We hypothesize that the IL-20 cytokine, and the IL cytokine subfamily more broadly, play a key role in CNS neuroinflammation by signaling through IL-20RB, induce VEGF activity, and enhance both BBB-permeability and CXCL12-mediated immune extravasation. To address this hypothesis, we actively immunized IL-20RB–/– mice and wild-type mice to induce experimental autoimmune encephalomyelitis (EAE) and found that IL-20RB–/– mice showed amelioration of disease progression compared to wild-type mice. Similarly, we passively immunized IL-20RB–/– mice and wild-type mice with myelin-reactive Th1 cells from either IL-20RB–/– and wild-type genotype. Host IL-20RB–/– mice showed lesser disease progression than wild-type mice, regardless of the myelin-reactive Th1 cells genotype. Using multianalyte bead-based immunoassay and ELISA, we found distinctive changes in levels of pro-inflammatory cytokines between IL-20RB–/– mice and wild-type mice at peak of EAE. We also found detectable levels of all cytokines of the IL-20 subfamily within CNS tissues and specific alteration to IL-20 subfamily cytokines IL-19, IL-20, and IL-24, expression levels. Immunolabeling of CNS region-specific microvessels confirmed IL-20RB protein at the spinal cord microvasculature and upregulation during EAE. Microvessels isolated from macaques CNS tissues also expressed IL-20RB. Moreover, we identified the expression of all IL-20 receptor subunits: IL-22 receptor subunit α-1 (IL-22RA1), IL-20RB, and IL-20 receptor subunit α (IL-20RA) in human CNS microvessels. Notably, human cerebral microvasculature endothelial cells (HCMEC/D3) treated with IL-1β showed augmented expression of the IL-20 receptor. Lastly, IL-20-treated HCMEC/D3 showed alterations on CXCL12 apicobasal polarity consistent with a neuroinflammatory status. This evidence suggests that IL-20 subfamily cytokines may signal at the BBB via IL-20RB, triggering neuroinflammation.

Study protocol of the PIMPI-project, a cohort study on acceptance, tolerability and immunogenicity of second trimester maternal pertussis immunization in relation to term and preterm infants

BackgroundMaternal immunization confers passive immunity to the fetus by transplacental antibody transfer. Infants may be better protected against pertussis if the mother received a diphtheriae, tetanus and acellular pertussis (Tdap) vaccination in the second trimester of pregnancy compared to the third trimester. This study evaluates IgG antibody concentrations in term and preterm infants at birth and 2 months after birth after maternal Tdap-vaccination between 200 and 240 w of gestation vs third trimester Tdap-vaccination. Further aims are assessing the determinants that underlie acceptance of second trimester maternal Tdap-vaccination as well as the tolerability of vaccination.MethodsThis prospective cohort study consists of two parts. In the acceptance part, pregnant women complete a questionnaire on determinants that underlie acceptance of a second trimester Tdap-vaccination, which is offered subsequently between 200 and 240 w of gestation. Vaccinated women complete an additional questionnaire on vaccination tolerability. Vaccinated women may also participate in the immunogenicity part, in which blood is drawn from mother at delivery and from infant at birth and 2 months after birth. Women are also eligible for the immunogenicity part if they received a Tdap-vaccination between 200 and 240 w of gestation via the national immunization program and get hospitalized for an imminent preterm delivery. Blood sampling continues until 60 term and 60 preterm mother-infant-pairs have been included. Pertussis-specific IgG antibody concentrations are determined in serum using a fluorescent bead-based multiplex immunoassay. For term infants, non-inferiority in IgG antibody concentrations against pertussis toxin (anti-PT) will be assessed referred to a historical control group in which mothers were Tdap-vaccinated between 300 and 320 w of gestation. For preterm infants, non-inferiority of anti-PT IgG concentrations is referred to as 85% of infants having ≥ 20 international units/mL at 2 months after birth.DiscussionThis study investigates acceptance, tolerability and immunogenicity regarding maternal Tdap-immunization between 200 and 240 w of gestation. Its results provide insight into the effects of second trimester Tdap-vaccination on IgG antibody concentrations in term and preterm infants before primary infant vaccinations. Results on acceptance and tolerability guide antenatal care providers in communication with pregnant women and maintain the safety of second trimester Tdap-vaccination.Trial registration: EU Clinical Trials Register, 2018-002976-41, retrospectively registered 24 July 2019, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2018-002976-41.

Relationship between Circulating Galectin-3, Systemic Inflammation, and Protein-Energy Wasting in Chronic Hemodialysis Patients.

Galectin-3 reportedly participates in the inflammatory process that causes insulin resistance in the target tissues. However, the role of high plasma galectin-3 levels as an indicator of protein-energy wasting (PEW) in patients undergoing maintenance hemodialysis remains unclear. This study included 240 hemodialysis patients (64.5 [55.3−74.0] years, 35.8% women) from a tertiary medical center. A baseline assessment of demographic and clinical data, biochemical parameters, and body composition was conducted. Plasma galectin-3 and other biomarkers were measured using a multiplex bead-based immunoassay. Participants were then divided into two subgroups depending on the median value of plasma galectin-3. Malnutrition was identified using the geriatric nutritional risk index (GNRI) and the criteria of the International Society of Renal Nutrition and Metabolism. Independent risk factors for elevated plasma galectin-3 and malnutrition were identified by multivariate logistic regression. The high galectin-3 group was more likely to be older, have lower lean tissue mass and GNRI scores, be diagnosed with PEW, dialyze through a tunneled catheter, and have higher circulating IL-6, TNF-α, and MCP-1 concentrations than the low galectin-3 group. After multivariate adjustment, only low mean arterial pressure, dialyzing with tunneled cuffed catheters, and elevated systemic inflammatory markers correlated with high galectin-3 levels. Plasma galectin-3 concentrations also increased significantly in hemodialysis patients with PEW. However, compared with other commonly used nutritional indicators, galectin-3 did not show superiority in predicting PEW. Although the plasma galectin-3 levels correlated with PEW severity, this correlation disappeared after adjustment for potential confounding variables (OR, 1.000; 95% CI, 0.999–1.001). In conclusion, plasma galectin-3 is a valuable biomarker for systemic inflammation but is less prominent for PEW in patients with maintenance hemodialysis. Further identification of novel biomarkers is required to detect patients at risk for malnutrition and implement appropriate interventions.

Circulation of Bordetella pertussis in the Caribbean Netherlands: a population-based seroepidemiological study

ObjectivesPertussis is a respiratory infectious disease caused by Bordetella pertussis. In the Caribbean Netherlands (CN), comprising the islands Bonaire, St Eustatius, and Saba, registration of cases is mandatory for disease surveillance. However, insufficient laboratory facilities hamper case confirmation, and circulation persists. The aim of this seroepidemiological study was to gain insight into B. pertussis circulation in CN, and to investigate what factors contribute to the risk of infection. MethodsBlood samples and questionnaires were collected for 1829 participants aged 0–90 years. Concentrations of B. pertussis toxin-specific IgG antibodies (anti-Pt) were determined using a bead-based immunoassay to indicate infections within the previous 12 months (based on anti-Pt ≥ 50 IU/mL) in participants without detectable vaccine-induced humoral immunity. Risk factors for recent infection were analyzed using logistic regression models. ResultsAn estimated 8.2% (95% CI 6.6–10.1) of CN residents aged ≥ 9 years were found to have been recently infected by B. pertussis. Risk factors for a recent infection were age 12–29 years (13.8–14.6%) and Dutch Caribbean or Surinamese origin (10.7%). ConclusionsB. pertussis infections occur frequently among CN residents aged ≥ 9 years, although few clinical pertussis cases are reported. Transmission to vulnerable individuals seems likely and should be taken into account in optimizing vaccination programs.

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.