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Objectives The present study aimed to investigate the distribution of lymphocyte subsets and cytokines expression in the peripheral blood of bitumen fumes-exposed workers. Methods In this study, 129 workers from molding and roasting workshops were recruited as the exposed group and 99 office and quality inspection staff were chosen as the control. The polycyclic aromatic hydrocarbons (PAHs) levels of bitumen fumes in individual and fixed-point air samples and the urinary levels of 1-hydroxypyrene (1-OH-P), 1-hydroxynaphthols (1-OH-N) and 2-hydroxynaphthols (2-OH-N) in workers were measured using High Performance Liquid Chromatography. The lymphocyte subsets and serum cytokines concentrations were analyzed by flow cytometry and cytometric bead array, respectively. Results The median values of PAHs were 0.08 mg/m3 for permissible concentration-time weighted average and 0.12 mg/m3 for permissible concentration-short term exposure (PC-STEL) in molding and roasting workshops, which were higher than that in the control area (< 0.01 mg/m3). Multivariate linear regression models were used to adjust for influential covariates, including age, gender, work age, smoking status, and alcohol consumptions. After adjusting for these covariates, we compared levels of urinary PAHs metabolites, the percentages of lymphocyte subsets, and serum cytokines concentrations between the two groups. The 1-OH-P, 1-OH-N, and 2-OH-N levels in the urine of bitumen fumes exposed workers were significantly higher than that in the controls (P < 0.05). Compared with the control group, the percentage of the natural killer (NK) cell (CD56+ cell) was significantly increased in the exposed group (P < 0.001). There was a significant decrease in the percentages of CD3+ T cell, CD4+ T cell, and CD8+ T cell in the exposed group compared to the control (P < 0.001). The serum levels of interleukin-1β (IL-1β) and IL-6 in bitumen fumes exposed workers were significantly higher than that of the controls (P < 0.05). Moreover, positive correlations were observed between the serum levels of IL-1β, IL-6, and urinary 1-OH-P levels in bitumen fumes-exposed workers, respectively (P < 0.05). There were no significant differences in the serum levels of IL-8, tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-1β (MIP-1β) and monocyte chemotactic protein-1 (MCP-1) between the exposed group and the control group (P > 0.05). Conclusion Our study suggested that low dose of bitumen fumes exposure could decrease the percentage of T cell, increase the percentage of NK cell and stimulate the release of serum IL-1β and IL-6 in the peripheral blood of exposed workers. The serum levels of IL-1β and IL-6 were positive correlated with the urinary 1-OH-P levels in bitumen fumes exposed workers. These results may inform the search for potential effective biomarkers and provide evidences for early health monitoring in workers occupationally exposed to bitumen fumes.

Background To understand the clinical, bacterial, and host characteristics associated with recurrent Staphylococcus aureus bacteremia (R-SAB), patients with R-SAB were compared to contemporaneous patients with a single episode of SAB (S-SAB). Methods All SAB isolates underwent spa genotyping. All isolates from R-SAB patients underwent pulsed-field gel electrophoresis (PFGE). PFGE-indistinguishable pairs from 40 patients underwent whole genome sequencing (WGS). Acute phase plasma from R-SAB and S-SAB patients was matched 1:1 for age, race, sex, and bacterial genotype, and underwent cytokine quantification using 25-analyte multiplex bead array. Results R-SAB occurred in 69 (9.1%) of the 756 study patients. Of the 69 patients, 30 experienced relapse (43.5%) and 39 reinfection (56.5%). Age, race, hemodialysis dependence, presence of foreign body, methicillin-resistant Staphyloccus aureus, and persistent bacteremia were individually associated with likelihood of recurrence. Multivariate risk modeling revealed that black hemodialysis patients were nearly 2 times more likely (odds ratio [OR] = 9.652 [95% confidence interval [CI], 5.402–17.418]) than white hemodialysis patients (OR = 4.53 [95% CI, 1.696–10.879]) to experience R-SAB. WGS confirmed PFGE interpretations in all cases. Median RANTES (regulated on activation, normal T cell expressed and secreted) levels in acute phase plasma from the initial episode of SAB were higher in R-SAB than in matched S-SAB controls (P = .0053, false discovery rate Conclusion This study identified several risk factors for R-SAB. The largest risk for R-SAB is among black hemodialysis patients. Higher RANTES levels in R-SAB compared to matched controls warrants further study.

Background Meat fraud and adulteration incidents occur frequently in almost all regions of the globe, especially with the increase in the world's population. To ensure the authenticity of meat products, we developed a 10-plex xMAP assay to simultaneously detect ten animal materials: bovine, caprine, poultry, swine, donkey, deer, horse, dog, fox and mink. Results This method was investigated by analyzing DNA extracts from raw muscle, muscle mixtures, meat products and animal feeds. Our results indicated that the species of interest can be identified, differentiated and detected down to 1 g kg-1 in binary mixtures or 0.01-0.001 ng of genomic DNA from specific species. Testing of 125 commercial samples showed a 97.4% coincidence rate with the method used in routine testing in our lab. Conclusion These results indicated that the method established in this study could detect ten animal materials simultaneously within 3 h, which provides a new, useful tool for animal ingredient analysis in meat products and animal feeds. © 2019 Society of Chemical Industry.

Spatial and molecular characteristics determine tissue function, yet high-resolution methods to capture both concurrently are lacking. Here, we developed High-Definition Spatial Transcriptomics (HDST), which captures RNA from histological tissue sections on a dense spatially barcoded bead array. Each experiment recovers several hundred thousand transcript-coupled spatial barcodes at 2μm resolution, as demonstrated in mouse brain and primary breast cancer. This opens the way to high-resolution spatial analysis of cells and tissues., Editorial summary A dense, spatially-barcoded bead array captures RNA from histological tissue sections for spatially-resolved gene expression analysis.

Highlights • NxTAG chemistry offers an easy workflow, low hands-on-time and scalable throughput. • All post-extraction assay steps take place in a single, closed tube. • Clinical performance of NxTAG RPP is comparable to that of the xTAG RVP assay. • Analytical performance is also similar or better than xTAG and xTAG FAST assays., The Luminex® NxTAG® Respiratory Pathogen Panel (NxTAG RPP) is an IVD-cleared assay for the simultaneous detection and identification of nucleic acids from 18 respiratory viruses and 2 (or 3 outside of the U.S.) atypical bacterial pathogens in nasopharyngeal swabs. Its scalability allows concurrent testing of up to 96 samples in a single batch. Nucleic acid extracted from 200 µL of raw specimen using the easyMAG® extractor is added directly to pre-plated, lyophilized bead reagents (LBRs), where multiplexed RT-PCR and hybridization to MagPlex-TAG™ microspheres occurs within a sealed reaction well using a single cycling program. Data acquisition is done on the MAGPIX® instrument which reads and sorts the reaction products directly from the sealed well following transfer of the assay plate from the thermal cycler. NxTAG is the newest innovation in bead-based nucleic acid chemistry developed by Luminex. Here we provide the detailed assay protocol and present data which describe the clinical and analytical performance characteristics of NxTAG RPP.

While human immunodeficiency virus (HIV) infection has become a treatable disease with the development of combination antiretroviral therapy (cART), chronic inflammation that affects the central nervous system and other organs is still common. Reliable methods are needed to study HIV-associated inflammatory biomarkers. In this study involving both plasma and cerebrospinal fluid (CSF), we compared multiplex bead array (MBA) to a relatively new technology based on microfluidics and glass nanoreactor (GNR) technology for the measurement of three commonly studied markers from HIV-infected individuals. We found that results correlated between the two platforms for MCP-1 in both fluids as well as for plasma TNFα (all p

Liquid biopsy, based on the molecular information extracted from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), offers the possibility to characterize the evolution of a solid tumor in real time and is highly important for diagnostic and therapeutic purposes. The aim of the present study was the development and validation of a novel liquid bead array methodology for the molecular characterization of CTCs and its application in breast cancer. In the present study we developed and evaluated a multiplex polymerase chain reaction (PCR)-coupled liquid bead array (MLBA) assay for studying simultaneously the expression of 14 genes in CTCs. The 14-gene MLBA assay is characterized by high analytical specificity, sensitivity, and reproducibility. The analytical performance of the 14-gene MLBA assay was compared with a commercially available test (AdnaTest BreastCancer, Qiagen, Germany) and our previously described multiplex quantitative reverse transcription PCR (RT-qPCR) assays. The developed assay has the potential to be further expanded in order to include up to 100 gene targets. The assay is highly specific for each target gene and is not affected by the numerous primers and probes used for multiplexing; hence, it constitutes a sample-, cost-, and time-saving analysis.

Systematic exploration of the dynamic human plasma proteome enables the discovery of novel protein biomarkers. Using state-of-the-art technologies holds the promise to facilitate a better diagnosis and risk prediction of diseases. Cardiovascular disease (CVD) pathophysiology is characterized for unbalancing of processes such as vascular inflammation, endothelial dysfunction, or lipid profiles among others. Such processes have a direct impact on the dynamic and complex composition of blood and hence the plasma proteome. Therefore, the study of the plasma proteome comprises an excellent exploratory source of biomarker research particularly for CVD. We describe the protocol for performing the discovery of protein biomarker candidates using the suspension bead array technology. The process does not require depletion steps to remove abundant proteins and consumes only a few microliters of sample from the body fluid of interest. The approach is scalable to measure many analytes as well as large numbers of samples. Moreover, we describe a bead-assisted antibody-labeling process that helps to develop quantitative assays for validation purposes and facilitate the translation of the identified candidates into clinical studies.

The flow cytometry-based multiplex bead array is an advanced technology using antibody-conjugated multiplex beads to detect soluble targets in a liquid phase. This technology has been widely used for detection of soluble analytes like cytokines, chemokines, allergens, viral antigens, and cancer markers. RayPlex® Multiplex Beads Antibody Array series are developed by RayBiotech Life, Inc. to quantitatively detect a wide range of analytes with high sensitivity to meet increasing need of research and diagnosis.

The use of biologic-based therapeutics has revolutionized our ability to treat complex diseases such as cancer- and autoimmune-related disorders. Biologic-based therapeutics are known to generate anti-drug immune responses or immunogenicity in clinical patients which can lead to altered pharmacokinetics, decreased drug efficacy, and unwanted adverse clinical events. Assays designed to detect and assess anti-drug immune responses are used to help monitor patients and improve drug safety. Utilizing a tiered approach, screening assays are developed first to identify patients that are potentially positive for anti-drug-specific antibodies. Patients that screen positive are subjected to additional tiers of testing that include a confirmation assay to confirm the presence of expected anti-drug-specific antibodies, a titer assay to assess relative levels of anti-drug-specific antibodies, and, depending on the drug’s mechanism of action or concerns of adverse clinical reactions, further characterization such as drug neutralization and anti-drug antibody isotyping. This tiered approach can prove to be detrimental to clinical samples from exposure to multiple cycles of testing, freeze thaws, and repeated handling by lab personnel. Multiplexing some of these assays together may streamline the characterization of anti-drug immune responses and help reduce the repeated usage of clinical samples. In this study, we combined a screening assay and anti-drug isotyping assays into one multiplexed assay using the Luminex® xMAP® Technology. The multiplexed assay was developed and validated to meet the FDA recommended guidelines for immunogenicity assessments. These results show that multiplexed assays perform comparably to industry standards. This study should encourage labs to explore the use of multiplexing immunogenicity assays to characterize anti-drug antibody responses quickly, with less repeat testing and reduced sample handling.

A detailed understanding of the adaptive host response to SARS-CoV-2 infection in humans is urgently needed, as questions abound regarding serologic responses and the development of antibody-mediated immunity against COVID-19. We developed a highly sensitive, high-throughput, and efficient assay using liquid bead array technology. We observed advantages over traditional indirect ELISA for the detection and quantification of binding IgG against the receptor binding domain (RBD) of SARS-CoV-2. Using this sensitive and highly reproducible assay, we sought to determine whether the severity of COVID-19 symptoms is correlated with SARS-CoV-2 binding IgG level. We measured SARS-CoV-2 RBD IgG levels from 67 subjects who had recovered from PCR-confirmed, symptomatic COVID-19. Samples were obtained approximately 6 weeks post-symptom onset. We found that COVID-19 symptom severity, assessed on an 8-point severity scale, strongly correlated with RBD IgG level (p

Background In recent years, there has been a significant decline in the demand for cord blood units (CBUs). This trend has led to cord blood banks (CBBs) exploring complementary uses of CBUs in order to exploit the full potential of this unique, valuable, and readily available product. The aim of this study was to establish standardized protocols for the preparation of four cord blood (CB) derivatives: plasma (CB-P), serum (CB-S), induced-serum (CB-IS) and growth factor-rich plasma (GFRP); to measure their respective growth-factor content and their potential as cell culture supplements, and finally, to identify the characteristics of each derivative beyond their growth-factor composition, in the context of optimizing CBBs resources. Study design and method To this end, CB plasma and serum were prepared and their concentrations for ten growth factors (TGF-β1, TGF-β2, TGF-β3, EGF, HGF, PDGF-BB, VEGF-A, VEGF-D, IGF-I, IGF-II) were determined using a multiplex bead array, and compared to adult plasma and serum. Protocols for the preparation of CB-IS and GFRP with reverse anticoagulation using CaCl2·2H2O were also established, and all four derivatives were compared for their EGF and TGF-β1 content by enzyme-linked immunosorbent assay (ELISA), and also for their cell culture supplement capacity. Results CB plasma was shown to be richer than adult plasma for TGF-β2 and TGF-β3, with a lower concentration of IGF-I and IGF-II. CB serum was shown to be richer than adult serum for TGF-β2, TGF-β3, EGF, HGF, PDGF-BB, and VEGF-A and D, and poorer in IGF-I and II. The measure of EGF and TGF-β1 concentrations has shown that CB serum is the most concentrated (1874 pg/ml and 41 094 pg/ml) of the CB derivatives, followed by induced-serum (405 pg/ml and 16 735 pg/ml), growth factor-rich plasma (131 and 7947 pg/ml) and plasma (8 and 2845 pg/ml). All four CB derivatives were shown to be superior to FBS in sustaining cell growth at low doses. Conclusion Our study shows that four CB derivatives can be easily prepared and pooled to provide significant volume of products that vary in their growth factor composition. A cord blood bank interested in introducing such manufacturing will need to evaluate the financial and processing characteristics of each derivative. The use of standardized manufacturing protocols such as the ones we suggest could help research initiatives exploring the potential therapeutic uses of such rich and high-quality starting material.

Quantitative assays that measure immune response to pneumococcal vaccines are not only important for the evaluation of vaccine immunogenicity and efficacy, but are also utilized in the clinical diagnosis of immune deficiency syndromes. Analytical methods have progressed in order to meet changing demands in both of these areas, from early methods to ELISA, and most recently multiplex bead array assays and opsonophagocytosis assays (OPA). It is necessary to understand the evolution of such techniques and the criteria for their interpretation in order to better inform the application of currently available methods, and to guide future investigation into assay development.

Cytokines are essential to maintain and coordinate the correct activity of immune cells during human pregnancy. IL-17 is a pro-inflammatory cytokine that induces the expression of many inflammatory mediators. The aim of this study was to compare the levels of Th1, Th2 and Th17 cytokines of women ongoing normal pregnancy with those found in women who suffered spontaneous abortion. IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ peripheral blood levels were measured in women who suffered spontaneous abortion (n = 13, blood collected up to 24 h after abortion), and were compared with healthy successful pregnancies (n = 16). Cytokine levels were measured using a cytometric bead array (CBA analysis). Similar cytokine levels were observed between spontaneous abortion and healthy pregnant women excepted to IL-17, which levels were increased in the healthy pregnant women (p = 0.0232). Our results show high IL-17 levels in the peripheral blood of women at late stages of healthy pregnancy, although low IL-17 levels were detected in the peripheral blood of women just after spontaneous abortion. In line with recent studies, this finding highlights IL-17 as a regulatory cytokine essential to the maintenance of a successful pregnancy.

Aim: The aim of this study was to validate the analytical performance of a combinatorial pharmacogenomics test designed to aid in the appropriate medication selection for neuropsychiatric conditions. Materials & methods: Genomic DNA was isolated from buccal swabs. Twelve genes (65 variants/alleles) associated with psychotropic medication metabolism, side effects, and mechanisms of actions were evaluated by bead array, MALDI-TOF mass spectrometry, and/or capillary electrophoresis methods (GeneSight Psychotropic, Assurex Health, Inc.). Results: The combinatorial pharmacogenomics test has a dynamic range of 2.5–20 ng/μl of input genomic DNA, with comparable performance for all assays included in the test. Both the precision and accuracy of the test were >99.9%, with individual gene components between 99.4 and 100%. Conclusion: This study demonstrates that the combinatorial pharmacogenomics test is robust and reproducible, making it suitable for clinical use.

Shigeo Yoshida,1 Yoshiyuki Kobayashi,1 Shintaro Nakao,1 Yukio Sassa,1,2 Toshio Hisatomi,1 Yasuhiro Ikeda,1 Yuji Oshima,1 Toshihiro Kono,2 Tatsuro Ishibashi,1 Koh-hei Sonoda1 1Department of Ophthalmology, Kyushu University Graduate School of Medical Sciences, Fukuoka, 2Fukuoka University Chikushi Hospital, Chikushino, Japan Background: Pars plana vitrectomy is the only treatment for advanced proliferative diabetic retinopathy (PDR). However, vitrectomy is not always successful despite current progress in vitreoretinal surgical techniques. The aim of our study was to investigate whether the vitreal concentrations of MCP-1, IL-6, IL-8, and VEGF are elevated after unsuccessful vitrectomy in patients with PDR and to investigate whether the altered levels of these cytokines are associated with the cause for the reoperation. Patients and methods: Vitreous samples were collected from 263 eyes of 233 patients: PDR (n=129 eyes), proliferative vitreoretinopathy (PVR; n=24 eyes) and nondiabetic controls (n=110 eyes) prior to vitrectomy. Vitreous samples were also collected from 14 eyes of 14 patients with PDR before vitrectomy and from the same 14 eyes before a second vitrectomy for reoperation. The levels of MCP-1, IL-6, IL-8, and VEGF were measured by flow cytometry using a cytometric bead array (CBA) assay. Results: The mean concentrations of vitreal MCP-1, IL-6, IL-8, and VEGF were significantly higher in patients with PDR and PVR (P

The co-administration of probiotics with inhaled steroids has been suggested as a potential treatment for recurrent wheezing in infants. This study aimed to elucidate if probiotics supplementation can improve the clinical parameters of wheezing and modify the expression of cytokines via T helper-1 and regulatory T cells. A randomised, double-blind, placebo-controlled clinical trial was conducted in 60 infants with recurrent wheezing treated with beclomethasone receiving a mixture of probiotics (kefir) or placebo daily for 8 weeks. IL-10, IL-12 and IFN-γ levels were evaluated by cytometric bead array (CBA) before and after 8 weeks of treatment for both groups. Probiotics mixture use was more likely to reduce the occurrence of wheezing during the intervention period and follow-up. There were significant differences in IL-10 and IL-12 levels in the blood cultures of infants who received the probiotics mixture. Thus, probiotics supplementation in infants treated for wheezing may have beneficial effect immunostimulatory.

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.

Accurate and sensitive quantification of a specific class of mycotoxins at trace levels in complex matrices with greener approaches is of significant importance. In this study, a green and economical protocol of magnetic microspheres-based cytometric bead array (CBA) assay on indirect competitive principle was developed for sensitive and rapid detection of ochratoxin A (OTA) in malts with a small number of standard and sample solutions. The protocol included the competition of OTA in malt samples and that covalently coupled on the surface of microspheres with its monoclonal antibodies, the separation and aggregation of the magnetic microspheres, and the fluorescence detection of fluorescein isothiocyanate labeled goat anti-mouse immunoglobulin G probes. The magnetic microspheres-based CBA assay allowed for ultralow limit of detection (0.025μgkg-1) for OTA and showed higher sensitivity compared with the common polystyrene beads-based CBA method. This is the first report on the magnetic microspheres-based CBA assay by using a simple and easy-to-operate magnetic separator for highly sensitive and rapid detection of OTA in complex malt samples. By consuming less solvent, time and cost, as well as fewer standard and samples, the developed green protocol expressed high potential for one-site real-time detection of trace components in complex matrices.

Tumor necrosis factor (TNF)-α is a key cytokine in the pathogenesis of dengue virus infection, and its accurate detection in several types of human samples is critical. The enzyme-linked immunosorbent assay (ELISA) is the gold standard for the detection of TNF-α, but multiplexed bead-based assays such as cytometric bead array (CBA) are now frequently used. Here, using ELISA and two CBAs commercially available, we measured TNF-α concentrations in plasma and serum from children with acute dengue virus infection and healthy controls. To evaluate the detection efficiency and factors affecting it, spiked recovery and immune complex dissociation assays were also performed. The levels of TNF-α evaluated by ELISA in paired serum and plasma samples from children with dengue positively correlated (rho = 0.99, p 0.0001). Children with dengue had higher levels of plasma TNF-α than those of healthy children (p = 0.004). The ELISA detected TNF-α in a higher number of plasma samples than the CBA (p 0.0001), and both methods only correlated when TNF-α was evaluated in buffer-based solutions but not in plasma, indicating the presence of a factor interfering with the detection of TNF-α in plasma. The recovery of several types of human recombinant TNF-α was dramatically decreased in plasma but not in tissue culture media (p ≤ 0.01), and this effect was similar in the plasma obtained from the children with dengue or the healthy controls. The dissociation of immune complexes did not improve TNF-α recovery. Dilution of the plasma samples increased the recovery of TNF-α, but at high concentrations of the cytokine. In short, plasma affects the efficiency of TNF-α detection, and this effect should be considered in the measurement of this cytokine.

A challenge in teaching immunology in the undergraduate laboratory is to encompass the many varied skills that need to be applied when performing an investigative study of such a complex area. It requires background knowledge, data analysis skills, critical thinking, and design capacities to include relevant controls and applications of particular techniques to answer a research question. It also requires strong technical skills. One such approach is to use inquiry-based learning which allows students a more proactive and integrative role in their learning. In one of our final year immunology units we have incorporated an inquiry-based exercise that runs across four 5-hour sessions. Students are given two cornerstone immunology techniques (ELISA and a flow cytometry-based cytokine bead array), which they use to formulate a study investigating inflammation. Stage one is to design the experiment with some guidance from teaching staff, stage two is to perform the experiment, and then finally students are required to analyze the data, apply appropriate statistics, and write a report outlining their findings. This approach provides students ownership of the process and allows them the opportunity to investigate a real-world problem rather than just attempting to obtain the expected "correct answer." Feedback from both students and staff has been positive with strong engagement and high quality reports produced.

We used a multiplexed suspension bead array immunoassay to map GPCR-receptor activity–modifying protein (RAMP) complexes., Receptor activity–modifying proteins (RAMPs) have been shown to modulate the functions of several G protein–coupled receptors (GPCRs), but potential direct interactions among the three known RAMPs and hundreds of GPCRs have never been investigated. Focusing mainly on the secretin-like family of GPCRs, we engineered epitope-tagged GPCRs and RAMPs, and developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized lysates. Using 64 antibodies raised against the native proteins and 4 antibodies targeting the epitope tags, we mapped the interactions among 23 GPCRs and 3 RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. The results provide a complete interactome of secretin-like GPCRs with RAMPs. The SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues.

A quantitative description of glyco-alteration/differences in diseases can lead to the development of a diagnostic agent for use in vitro to monitor the degree of change in target glycoproteins. Analytical systems have been developed along with the progress of omics-oriented technologies. For clinical implementation, their full automation is required with an apparatus that is simple to operate. Here, we report an automatic analysis system for quantitative characterization of glyco-alteration/differences that depends on the unique strategy of "bead arrays in a single tip." The alternative lectin array can obtain a minimum characterization of the glycan profile for nanogram quantities of an endogenous glycoprotein. A simple autopipetting robot produces the precise chemiluminescence detection of glycan-lectin interactions with a wide dynamic range that is superior to fluorescence-based lectin arrays. The tip-based array format enables automatic glycan profiling from sample pretreatment to detection with low variation and linear detection, which may facilitate the use of this lectin array in clinical practice.

The development of cross-reactive sensor arrays for volatile organics (electronic noses, e-noses) is an active area of research. In this manuscript, we present a new format for barcoded polymer sensor arrays based on porous polymer beads. An array of nine self-encoded polymers was analyzed by Raman spectroscopy before and after exposure to a series of volatile organic compounds, and the changes in the vibrational fingerprints of their polymers was recorded before and after exposure. Our results show that the spectroscopic changes experienced by the porous spectroscopically encoded beads after exposure to an analyte can be used to identify and classify the target analytes. To expedite this analysis, analyte-specific changes induced in the sensor arrays were transformed into a response pattern using multivariate data analysis. These studies established the barcoded bead array format as a potentially effective sensing element in e-nose devices. Devices such as these have the potential to advance personalized medicine, providing a platform for non-invasive, real-time volatile metabolite detection.

The plasma proteome offers a clinically useful window into human health and disease. With recent progress made on the development of highly multiplexed immunoassays with high sample throughput, a remaining need is to establish a pipeline for validating the individual proteins that build such bio-signatures by using targeted assays. In order to streamline such efforts, we developed a workflow to build dual binder sandwich immunoassays (SIA) and chose to evaluate this on proteins predicted to be secreted form cells and tissues. Utilizing the multiplexing capacities of the bead array technology, we first screened ~ 1,800 unique antibody pairs against 209 protein targets and collected data from dilution series of recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies from the Human Protein Atlas, we obtained dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets. For 22 protein assays, the longitudinal, inter-individual and technical performance was determined in a set of plasma samples collected from 18 healthy subjects every third month over one year. Lastly, we compared 14 of these assays with SIAs composed of other binders, proximity extension assays and affinity-free targeted mass spectrometry. Our workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, while the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.