Bladder cancer risk stratification with the Oncuria 10-plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms.

BACKGROUND: No single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories. METHODS: We compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow-based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. All three analyzers quantify fluorescence signals generated by the Oncuria assay. RESULTS: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically < 5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥ 1 analyte above the highest standard concentration, i.e., A1AT (n = 7/18), IL-8 (n = 5), and/or ANG (n = 2), while only one control sample registered an analyte (A1AT) above the highest standard concentration. CONCLUSION: Multiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment responsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.

Temperature impacts the bovine ex vivo immune response towards Mycoplasmopsis bovis.

Although cattle are the mammalian species with most global biomass associated with a huge impact on our planet, their immune system remains poorly understood. Notably, the bovine immune system has peculiarities such as an overrepresentation of γδ T cells that requires particular attention, specifically in an infectious context. In line of 3R principles, we developed an ex vivo platform to dissect host-pathogen interactions. The experimental design was based on two independent complementary readouts: firstly, a novel 12-14 color multiparameter flow cytometry assay measuring maturation (modulation of cell surface marker expression) and activation (intracellular cytokine detection) of monocytes, conventional and plasmacytoid dendritic cells, natural killer cells, γδ T cells, B and T cells; secondly, a multiplex immunoassay monitoring bovine chemokine and cytokine secretion levels. The experiments were conducted on fresh primary bovine blood cells exposed to Mycoplasmopsis bovis (M. bovis), a major bovine respiratory pathogen. Besides reaffirming the tight cooperation of the different primary blood cells, we also identified novel key players such as strong IFN-γ secreting NK cells, whose role was so far largely overlooked. Additionally, we compared the host-pathogen interactions at different temperatures, including commonly used 37 °C, ruminant body temperature (38-38.5 °C) and fever (≥ 39.5 °C). Strikingly, working under ruminant physiological temperature influenced the capacity of most immune cell subsets to respond to M. bovis compared to 37 °C. Under fever-like temperature conditions the immune response was impaired compared to physiological temperature. Our experimental approach, phenotypically delineating the bovine immune system provided a thorough vision of the immune response towards M. bovis and the influence of temperature towards that immune response.

Elucidating the molecular healing of intrabony defects following non-surgical periodontal therapy: A pilot study.

OBJECTIVE: To elucidate the molecular healing of intrabony defects following non-surgical periodontal therapy (NSPT) using gingival crevicular fluid (GCF). BACKGROUND DATA: Currently limited information is available regarding the GCF of intrabony defects and the change in biomarker levels in the GCF at early time points following treatment interventions. METHODS: Twenty-one patients (Periodontitis Stage III or IV) who have received NSPT, contributing one intrabony defect and one healthy site were included in this study. GCF sampling was performed at baseline, 1 day, 5 days and 3 months after NSPT. Multiplex bead immunoassays allowed the profiling of GCF for 27 markers, associated with inflammation and repair/regeneration. A mixed effects model with Bonferroni correction for multiple comparisons was employed to compare the changes in the levels of GCF markers over time. RESULTS: Following NSPT, changes were observed for several GCF markers, marked by significant increases 1 day post-intervention, before returning to baseline levels by 3 months. Specifically, GCF concentrations of IL-2, IL-4, IL-6, IL-8, MMP-1, MMP-3, TIMP-1 and FGFb significantly increased 1 day after NSPT. Signs of activation of cellular senescence were observed 1 day following treatment of intrabony defects, rapidly regressing by 5 days. CONCLUSION: Significant molecular changes are observed as early as 1 day following NSPT in intrabony defects, along with activation of cellular senescence.

Soft-Tissue Phenotype as a Risk Indicator of Peri-Implantitis and Peri-Implant Soft-Tissue Dehiscence-A Cross-Sectional Study.

AIM: To investigate the association, as well as to characterize the associated panel of pro- and anti-inflammatory markers, between the different components of the peri-implant phenotype and the presence of peri-implantitis/peri-implant soft-tissue dehiscence (PISTD). MATERIALS AND METHODS: A total of 324 implants in 112 patients were included. The following components of the peri-implant phenotype were clinically measured through the use of a manual periodontal probe or a digital calliper: keratinized mucosa width (PIKM-W), mucosal thickness (MT), attached mucosa (AM) and vestibulum depth (VD). The presence of peri-implantitis and PISTD was assessed through clinical and radiographic examination. Mixed-models logistic regression analyses were performed to analyse the association between peri-implant phenotype and the presence of peri-implantitis or PISTD, adjusting for relevant confounders. Multiplex immunoassays were employed to evaluate the peri-implant crevicular fluid levels of a panel of pro- and anti-inflammatory markers. RESULTS: Peri-implant health, peri-implant mucositis and peri-implantitis were diagnosed in 36.6%, 21.4% and 42% of the patients (classified according to their worst implant) and 35.2%, 34.3%, and 30.5% of the implants, respectively. In the multi-level multiple regression model, the absence of PIKM-W (odds ratio [OR] = 9.24; 95% CI: 2.73-31.28), the absence of attached mucosa (OR = 19.58; 95% CI: 6.12-62.56) and a reduced (<4 mm) vestibulum depth (OR = 2.61; 95% CI: 1.05-6.48) were associated with peri-implantitis. Similarly, the absence of PIKM-W (OR = 6.32; 95% CI: 1.67-23.83), a thin (<2 mm) mucosa (OR = 157.75; 95% CI: 14.06-1769.9) and a reduced vestibulum depth (OR = 3.32; 95% CI: 1.02-10.84) were associated with the presence of PISTD. Implants with PIKM-W = 0 mm showed statistically significantly higher levels of interferon-γ in both regular (≥2 maintenance/year) and irregular (<2 maintenance/year) compliers (p = 0.046 and p = 0.012). In irregular compliers, the absence of PIKM-W was also associated with statistically significantly higher levels of interleukin (IL)-1ß and IL-21 (p = 0.016, p = 0.046). These associations were independent of the effect of relevant confounders (e.g., plaque, compliance with maintenance, etc.). CONCLUSIONS: Within their limits, the present findings indicate that (a) peri-implant soft-tissue phenotype appears to be associated with the presence of peri-implantitis and PISTD, and (b) in the absence of PIKM-W, the inflammatory response seems to be dysregulated and the soft-tissue remodelling up-regulated.

VCAM-1 complements CA-125 in detecting recurrent ovarian cancer.

BACKGROUND: Close to three-quarters of ovarian cancer cases are frequently diagnosed at an advanced stage, with more than 70% of them failing to respond to primary therapy and relapsing within 5 years. There is an urgent need to identify strategies for early detection of ovarian cancer recurrence, which may lead to earlier intervention and better outcomes. METHODS: A customized magnetic bead-based 8-plex immunoassay was evaluated using a Bio-Plex 200 Suspension Array System. Target protein levels were analyzed in sera from 58 patients diagnosed with advanced ovarian cancer (including 34 primary and 24 recurrent tumors) and 46 healthy controls. The clinical performance of these biomarkers was evaluated individually and in combination for their ability to detect recurrent ovarian cancer. RESULTS: An 8-plex immunoassay was evaluated with high analytical performance suitable for biomarker validation studies. Logistic regression modeling selected a two-marker panel of CA-125 and VCAM-1 that improved the performance of CA-125 alone in detecting recurrent ovarian cancer (AUC: 0.813 versus 0.700). At a fixed specificity of 83%, the two-marker panel significantly improved sensitivity in separating primary from recurrent tumors (70.8% versus 37.5%, P = 0.004), demonstrating that VCAM-1 was significantly complementary to CA-125 in detecting recurrent ovarian cancer. CONCLUSIONS: A two-marker panel of CA-125 and VCAM-1 showed strong diagnostic performance and improvement over the use of CA-125 alone in detecting recurrent ovarian cancer. The experimental results warrant further clinical validation to determine their role in the early detection of recurrent ovarian cancer.

Cytoskeletal Alteration Is an Early Cellular Response in Pulmonary Epithelium Infected with Aspergillus fumigatus Rather than Scedosporium apiospermum.

Invasive aspergillosis and scedosporiosis are life-threatening fungal infections with similar clinical manifestations in immunocompromised patients. Contrarily, Scedosporium apiospermum is susceptible to some azole derivative but often resistant to amphotericin B. Histopathological examination alone cannot diagnose these two fungal species. Pathogenesis studies could contribute to explore candidate protein markers for new diagnosis and treatment methods leading to a decrease in mortality. In the present study, proteomics was conducted to identify significantly altered proteins in A549 cells infected with or without Aspergillus fumigatus and S. apiospermum as measured at initial invasion. Protein validation was performed with immunogold labelling alongside immunohistochemical techniques in infected A549 cells and lungs from murine models. Further, cytokine production was measured, using the Bio-Plex-Multiplex immunoassay. The cytoskeletal proteins HSPA9, PA2G4, VAT1, PSMA2, PEX1, PTGES3, KRT1, KRT9, CLIP1 and CLEC20A were mainly changed during A. fumigatus infection, while the immunologically activated proteins WNT7A, GAPDH and ANXA2 were principally altered during S. apiospermum infection. These proteins are involved in fungal internalisation and structural destruction leading to pulmonary disorders. Interleukin (IL)-21, IL-1α, IL-22, IL-2, IL-8, IL-12, IL-17A, interferon-γ and tumour necrosis factor-α were upregulated in both aspergillosis and scedosporiosis, although more predominately in the latter, in accordance with chitin synthase-1 and matrix metalloproteinase levels. Our results demonstrated that during invasion, A. fumigatus primarily altered host cellular integrity, whereas S. apiospermum chiefly induced and extensively modulated host immune responses.

Multiplexed flow cytometric approach for detection of anti-SARS-CoV-2 IgG, IgM and IgA using beads covalently coupled to the nucleocapsid protein.

Flow cytometry has emerged as a promising technique for detection of SARS-CoV-2 antibodies. In this study, we developed an innovative strategy for simultaneous detection of immunoglobulin G (IgG), IgM and IgA. The SARS-CoV-2 nucleocapsid protein was covalently bound to functional beads surface applying sulpho-SMCC chemistry. BUV395 anti-IgG, BB515 anti-IgM, biotinylated anti-IgA1/IgA2 and BV421 streptavidin were used as fluorophore conjugated secondary antibodies. Serum and antibodies reaction conditions were optimized for each antibody isotype detection and a multiplexed detection assay was developed. This new cell-free assay efficiently discriminate COVID-19 negative and positive samples. The simultaneous detection of IgG, IgM and IgA showed a sensitivity of 88·5-96·2% and specificity of 100%. This novel strategy opens a new avenue for flow cytometry-based diagnosis.

The inflammatory response and blood-spinal cord barrier integrity in traumatic spinal cord injury: a prospective pilot study.

PURPOSE: Triggering of inflammatory responses and disruption of blood-spinal cord barrier (BSCB) integrity are considered pivotal events in the pathophysiology of traumatic spinal cord injury (TSCI). Yet, these events are poorly understood and described in humans. This study aims to describe inflammatory responses and BSCB integrity in human TSCI. METHODS: Fifteen TSCI patients and fifteen non-TSCI patients were prospectively recruited from Aarhus University Hospital, Denmark. Peripheral blood (PB) and cerebrospinal fluid (CSF) were collected at median day 0 [IQR: 1], median day 9 [IQR: 2], and median day 148 [IQR: 49] after injury. PB and CSF were analyzed for immune cells by flow cytometry, cytokines by multiplex immunoassay, and BSCB integrity by IgG Index. RESULTS: Eleven TSCI patients completed follow-up. Results showed alterations in innate and adaptive immune cell counts over time. TSCI patients had significantly increased cytokine concentrations in CSF at the first and second follow-up, while only concentrations of interleukin (IL)-4, IL-8, and tumor necrosis factor-α remained significantly increased at the third follow-up. In PB, TSCI patients had significantly increased IL-6, IL-8, and IL-10 concentrations and significantly decreased interferon-γ concentrations at the first follow-up. Results further showed increased IgG Index indicative of BSCB disruption in seven TSCI patients at the first follow-up, five TSCI patients at the second follow-up, and two patients at the third follow-up. CONCLUSIONS: Our results suggest that TSCI mainly triggers innate inflammatory responses that resolves over time, although with some degree of non-resolving inflammation, particularly in CSF. Our results cannot confirm BSCB disruption in all TSCI patients.

Microfluidic synthesis of Janus-structured QD-encoded magnetic microbeads for multiplex immunoassay.

Uniform and monodisperse quantum dot (QD)-encoded magnetic microbeads with Janus structure were produced in a microfluidic device via photopolymerization. UV light through a microscope objective was used to solidify the microbeads which showed sharp interfaces and excellent magnetic responses. QDs with different emission peaks (450 nm for blue and 640 nm for red) were mixed at different ratios to provide three spectral codes. The QD-encoded microbeads can be distinguished by analyzing their fluorescent images in HSV color space. After hydrolysis of the anhydride group in alkaline solution, protein was immobilized on microbeads via activation of carboxyl groups using (1-ethyl-3(3-dimethylaminoprophyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). A microhole array in polydimethylsiloxane (PDMS) substrates with a specific size was fabricated to trap individual microbeads in a single microhole. The combination of Janus-structured QD-encoded magnetic microbeads and microhole arrays facilitates both flexibility, binding kinetics, sensitivity for suspension assay, and fluorescence mapping analysis for conventional biochips, thus providing a novel platform for multiplex bioanalysis. The capability of this integration for multiplex immunoassays was verified using three kinds of IgG and their corresponding anti-IgG. A detection limit of 0.07 ng/mL was achieved for human IgG, indicating practical applications in various fields.

Multiple biomarker approach for the diagnosis and therapy of rheumatoid arthritis.

The lack of specific clinical symptoms for patients in the early stage of rheumatoid arthritis (RA) has created strong interest in the laboratory diagnosis of RA. The main laboratory markers of RA, rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs), can be found in patients with other pathologies and in healthy donors. Even today, there is no single laboratory test that can diagnosis RA with high sensitivity and specificity. To improve the diagnosis and treatment of RA, alternative biomarkers, including 14-3-3η protein, connective tissue growth factor (CTGF), antibodies against PAD4, antibodies against BRAF, and anti-acetylated and anti-carbamylated protein antibodies have been studied extensively. The use of a multiple biomarker approach, the simultaneous measurement of a set of biomarkers, is an alternative strategy for the diagnosis of RA and for predicting the therapeutic effect of biological disease-modifying antirheumatic drugs (DMARDs). However, despite the large number of studies, only a few biomarker combinations have been validated and can be applied in clinical practice. In this article, results of studies focused on the multiple biomarker approach (both multiplex and combined single-analyte assays) to diagnose RA and to predict response to biological drug therapy are reviewed. Additionally, general factors limiting the use of multiplex analysis in RA diagnostics and therapy are discussed.

Diagnostic characterization of respiratory allergies by means of a multiplex immunoassay.

Allergic sensitization is commonly assessed in patients by performing the skin prick test (SPT) or determining specific immunoglobulin (IgE) levels in blood samples with the ImmunoCAP™ assay, which measures each allergen and sample separately. This paper explores the possibility to investigate respiratory allergies with a high throughput method, the Meso Scale Discovery (MSD) multiplex immunoassay, measuring IgE levels in low volumes of blood. The MSD multiplex immunoassay, developed and optimized with standards and allergens from Radim Diagnostics, was validated against the SPT and the ImmunoCAP assay. For 18 adults (15 respiratory allergy patients and three controls), blood collection and the SPT were performed within the same hour. Pearson correlations and Bland-Altman analysis showed high comparability of the MSD multiplex immunoassay with the SPT and the ImmunoCAP assay, except for house dust mite. The sensitivity of the MSD multiplexed assay was ≥78% for most allergens compared to the SPT and ImmunoCAP assay. Additionally, the specificity of the MSD multiplex immunoassay was ≥ 87% - the majority showing 100% specificity. Only the rye allergen had a low specificity when compared to the SPT, probably due to cross-reactivity. The reproducibility of the MSD multiplex immunoassay, assessed as intra- and interassay reproducibility and biological variability between different sampling moments, showed significantly high correlations (r = 0·943-1) for all tested subjects (apart from subject 13; r = 0·65-0·99). The MSD multiplex immunoassay is a reliable method to detect specific IgE levels against respiratory allergens in a multiplexed and high-throughput manner, using blood samples as small as from a finger prick.

High-throughput multiplex assays with mouse macrophages on pillar plate platforms.

It is challenging to rapidly identify immune responses that reflect the state and capability of immune cells due to complex heterogeneity of immune cells and their plasticity to pathogens and modulating molecules. Thus, high-throughput and easy-to-use cell culture and analysis platforms are highly desired for characterizing complex immune responses and elucidating their underlying mechanisms as well. In response to this need, we have developed a micropillar chip and a 384-pillar plate, printed mouse macrophage, RAW 264.7 cell line in alginate on the pillar plate platforms, and established multiplex cell-based assays to rapidly measure cell viability, expression of cell surface markers, and secretion of cytokines upon stimulation with model compound, lipopolysaccharide (LPS), as well as synthetic N-glycan polymers that mimic native glycoconjugates and could bind to lectin receptors on RAW 264.7 cells. Interestingly, changes in RAW 264.7 cell viability, expression levels of cell surface makers, and release of cytokines measured from the pillar plate platforms in the presence and absence of LPS were well correlated with those obtained from their counterpart, the 96-well plate with 2D-cultured macrophages. With this approach, we identified that α2,3-linked N-sialyllactose polymer has significant macrophage modulation activity among the N-glycan polymers tested. Therefore, we successfully demonstrated that our pillar plate platforms with 3D-cultured macrophages can streamline immune cell imaging and analysis in high throughput in response to compound stimulation. We envision that the pillar plate platforms could potentially be used for rapid characterization of immune cell responses and for screening immune cell-modulating molecules.

Biomarker levels in peri-implant crevicular fluid of healthy implants, untreated and non-surgically treated implants with peri-implantitis.

AIM: To compare biomarker levels in peri-implant crevicular fluid (PICF) of healthy implants with levels in PICF of implants with peri-implantitis (before and after non-surgical treatment). MATERIALS AND METHODS: Samples were taken from 20 healthy implants (n = 17 patients) and from 20 implants with peri-implantitis (n = 19 patients) before and 3 months after non-surgical treatment using the Airflow Master Piezon® (EMS). A Luminex™ assay was used to evaluate pro-inflammatory and anti-inflammatory cytokines IL-1ß, TNF-α, IL-6 and G-CSF, collagen degradation enzyme MMP-8, chemokines MCP-1 & MIP-1α/CCL3, bone markers OPG and sRANKL and interferon-γ. Clinical and radiographical characteristics were assessed. A Mann-Whitney U and Wilcoxon signed-rank test analysed between- and within-group differences. RESULTS: IL-1ß and MMP-8 levels were found significantly elevated in implants with peri-implantitis (p = .007; p = <.001, respectively). No difference in levels of TNF-α, IL-6, MCP-1 and MIP-1α/CCL3, OPG and G-CSF between healthy and diseased implants was found. Levels of sRANKL and INF-γ were under the level of detection. None of the biomarker levels improved after non-surgical therapy, and levels of IL-1ß and MMP-8 remained high. CONCLUSION: Implants diagnosed with peri-implantitis have higher levels of IL-1ß and MMP-8 in PICF compared to healthy implants. Non-surgical therapy did not influence the inflammatory immune response.

Evidence for Altered Phosphoinositide Signaling-Associated Molecules in the Postmortem Prefrontal Cortex of Patients with Schizophrenia.

Phosphoinositides (PIs) play important roles in the structure and function of the brain. Associations between PIs and the pathophysiology of schizophrenia have been studied. However, the significance of the PI metabolic pathway in the pathology of schizophrenia is unknown. We examined the expression of PI signaling-associated proteins in the postmortem brain of schizophrenia patients. Protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA, also known as PIK4A), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), protein kinase B (Akt), and glycogen synthase kinase 3ß (GSK3ß) were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays of the prefrontal cortex (PFC) of postmortem samples from 23 schizophrenia patients and 47 normal controls. We also examined the association between PIK4CA expression and its genetic variants in the same brain samples. PIK4CA expression was lower, whereas Akt expression was higher, in the PFC of schizophrenia patients than in that of controls; PIP5K1C, PTEN, and GSK3ß expression was not different. No single-nucleotide polymorphism significantly affected protein expression. We identified molecules involved in the pathology of schizophrenia via this lipid metabolic pathway. These results suggest that PIK4CA is involved in the mechanism underlying the pathogenesis of schizophrenia and is a potential novel therapeutic target.

Rapid Salivary IgG Antibody Screening for Hepatitis A.

Hepatitis A virus (HAV) is a common infection that is transmitted through the fecal-oral route, shed in the stool of infected individuals, and spread either by direct contact or by ingesting contaminated food or water. Each year, approximately 1.4 million acute cases are reported globally with a major risk factor for exposure being low household socioeconomic status. Recent trends show a decrease in anti-HAV antibodies in the general population, with concomitant increases in the numbers of HAV outbreaks. In line with a recreational water study, this effort aims to assess the prevalence of salivary IgG antibodies against HAV and subsequent incident infections (or immunoconversions) in visitors to a tropical beach impacted by a publicly owned treatment works (POTW). We applied a multiplex immunoassay to serially collected saliva samples gathered from study participants who recreated at Boquerón Beach, Puerto Rico. Analysis of assay results revealed an immunoprevalence rate of 16.17% for HAV with 1.43% of the cohort immunoconverting to HAV. Among those who immunoconverted, 10% reported chronic gastrointestinal symptoms and none experienced diarrhea. Tests on water samples indicated good water quality with low levels of fecal indicator bacteria; however, the collection and analysis of saliva samples afforded the ability to detect HAV infections in beachgoers. This rapid assay serves as a cost-effective tool for examining exposure to environmental pathogens and can provide critical information to policy makers, water quality experts, and risk assessment professionals seeking to improve and protect recreational water and public health.

Measuring antibody avidity to Plasmodium falciparum merozoite antigens using a multiplex immunoassay approach.

BACKGROUND: Antibodies (Ab) play a significant role in immunity to Plasmodium falciparum malaria. Usually, following repeated exposure to pathogens, affinity maturation and clonal selection take place, resulting in increased antibody avidity. However, some studies suggest affinity maturation may not occur to malaria antigens in endemic areas. Information on development of antibody avidity is confusing and conflicting, in part, because different techniques have been used to measure avidity. Today, bead-based multiplex immunoassays (MIA) are routinely used to simultaneously quantitate antibody levels to multiple antigens. This study evaluated the feasibility of developing an avidity MIA with 5 merozoite antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3) that uses a single chaotropic concentration. METHODS: The most common ELISA protocols that used the chaotropic reagents guanidine HCl (GdHCl), urea, and ammonium thiocyanate (NH4SCN) were adapted to a multiplex MIA format. Then, different concentrations of chaotropes and incubation times were compared and results were expressed as an Avidity Index (AI), i.e., percentage of antibody remaining bound in the presence of chaotrope. Experiments were conducted to (i) identify the assay with the widest range of AI (discriminatory power), (ii) determine the amount of chaotrope needed to release 50% of bound Ab using plasma from adults and infants, and (iii) evaluate assay repeatability. RESULTS: Overall, 4 M GdHCl and 8 M urea were weaker chaotropes than 3 M NH4SCN. For example, they failed to release significant amounts of Ab bound to MSP1-42 in adult plasma samples; whereas, a range of AI values was obtained with NH4SCN. Titration of NH4SCN revealed that 2 M NH4SCN gave the widest range of AI for the 5 antigens. Binding studies using plasma from 40 adults and 57 1-year old infants in Cameroon showed that 2.1 M ± 0.32 (mean ± SD) NH4SCN (adults) and 1.8 M ± 0.23 M (infants) released 50% of bound Ab from the merozoite antigens. CONCLUSIONS: An avidity MIA is feasible for the 5 merozoite antigens that uses a single concentration (2 M) of NH4SCN. The assay provides a simple method to quickly obtain information about Ab quantity and quality in the acquisition of immunity to malaria in endemic populations.

Cytokine and growth factor profile in endometriosis: a multiplex analysis of peritoneal fluid to assess diagnostic utility.

We aimed to assay cytokines and growth factors in peritoneal fluid samples from women with and without endometriosis to understand the inflammatory milieu, and assess their potential diagnostic utility. This cross-sectional study conducted at a tertiary care hospital included 54 women, aged 20-45 years, with regular menstrual history and undergoing diagnostic/therapeutic laparoscopy for infertility and/or pain. Peritoneal fluid samples were collected after insertion of trocar & laparoscope but prior to other surgical intervention. A multiplex immunoassay of 27 cytokines and growth factors was performed. The concentration of FGF2 and CSF3 were significantly lower in women with endometriosis than without endometriosis (p = .043 and .003, respectively). The levels of CCL2 and IL1RN were significantly higher in moderate-severe than in minimal-mild endometriosis (p = .038 and .043, respectively). Phase-specific comparison revealed that in proliferative phase, the levels of CSF2 and CSF3 were lower in women with endometriosis than without the disease (p = .047 and .013, respectively). The ROC curve analysis provided a cutoff value 0.78 and 0.76 for FGF2 and CSF3, respectively. Cytokines and growth factors such as FGF2, CSF3, CSF2, CCL2 and IL1RN seem to contribute to the pathogenesis of endometriosis and may have a potential utility for the diagnosis of endometriosis.

Simultaneous determination of gastric cancer biomarkers pepsinogen PGI/PGII using element tagged immunoassay coupled with inductively coupled plasma mass spectrometry detection.

OBJECTIVES: In this study, a new immunoassay for the simultaneous determination of pepsinogen I (PGI) and pepsinogen II (PGII) in serum based on element labeling strategy coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection was proposed. METHODS: The sandwich-type immunoassay was used to simultaneously detect PGI and PGII in serum. PGI and PGII were captured by anti-PGI and anti-PGII antibody immobilized on the magnetic beads and then banded with Eu3+ labeled anti-PGI detection antibody and Sm3+ labeled anti-PGII detection antibody, followed by ICP-MS detection. RESULTS: The linear correlation coefficient (R2 ) of PGI and PGII standard curves was .9938 and .9911, with the dynamic range of 0-200 ng/mL and 0-60 ng/mL, respectively. The limit of detection for PGI and PGII was 1.8 ng/mL and 0.3 ng/mL, respectively. The average recovery was 101.41% ± 6.74% for PGI and 101.47% ± 4.20% for PGII. Good correlations were obtained between the proposed method and CLIA (r = .9588 for PGI, r = .9853 for PGII). CONCLUSIONS: We established a mass spectrometry-based immunoassay for the simultaneous detection of PGI and PGII in a single analysis. The element tagged immunoassay coupled with ICP-MS detection has high sensitivity, accuracy, and specificity in clinical serum sample analysis.

Defining Metaniches in the Oral Cavity According to Their Microbial Composition and Cytokine Profile.

The human oral microbiota consists of over 700 widespread taxa colonizing the oral cavity in several anatomically diverse oral niches. Lately, sequencing of the 16S rRNA genes has become an acknowledged, culture-independent method to characterize the oral microbiota. However, only a small amount of data are available concerning microbial differences between oral niches in periodontal health and disease. In the context of periodontitis, the cytokine expression in the gingival crevicular fluid has been studied in detail, whereas little is known about the cytokine profile in hard and soft tissue biofilms. In order to characterize oral niches in periodontal health, the oral microbiota and cytokine pattern were analyzed at seven different sites (plaque (P), gingival crevicular fluid (GCF), saliva (S), tongue (T), hard palate (HP), cheek (C) and sublingual area (U)) of 20 young adults using next-generation sequencing and multiplex immunoassays. Site-specific microbial compositions were detected, which clustered into three distinct metaniches ("P-GCF", "S-T-HP" and "C-U") and were associated with niche-/metaniche-specific cytokine profiles. Our findings allow the definition of distinct metaniches according to their microbial composition, partly reflected by their cytokine profile, and provide new insights into microenvironmental similarities between anatomical diverse oral niches.

Minimally Invasive Saliva Testing to Monitor Norovirus Infection in Community Settings.

BACKGROUND: Norovirus is a leading cause of acute gastroenteritis worldwide. Routine norovirus diagnosis requires stool collection. The goal of this study was to develop and validate a noninvasive method to diagnose norovirus to complement stool diagnostics and to facilitate studies on transmission. METHODS: A multiplex immunoassay to measure salivary immunoglobulin G (IgG) responses to 5 common norovirus genotypes (GI.1, GII.2, GII.4, GII.6, and GII.17) was developed. The assay was validated using acute and convalescent saliva samples collected from Peruvian children <5 years of age with polymerase chain reaction (PCR)-diagnosed norovirus infections (n = 175) and controls (n = 32). The assay sensitivity and specificity were calculated to determine infection status based on fold rise of salivary norovirus genotype-specific IgG using norovirus genotype from stool as reference. RESULTS: The salivary assay detected recent norovirus infections and correctly assigned the infecting genotype. Sensitivity was 71% and specificity was 96% across the evaluated genotypes compared to PCR-diagnosed norovirus infection. CONCLUSIONS: This saliva-based assay will be a useful tool to monitor norovirus transmission in high-risk settings such as daycare centers or hospitals. Cross-reactivity is limited between the tested genotypes, which represent the most commonly circulating genotypes.