Effectiveness of Water-Based Exercise in Patients with Chronic Obstructive Pulmonary Disease: Systematic Review and Meta-Analysis.

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disease that, due to dyspnea, decreases patients' physical function and quality of life. The aim of the research was to evaluate the effectiveness of water-based exercise (WE) in improving functional capacity and respiratory muscle strength in patients with COPD. It consisted of a systematic review and meta-analysis of eight randomized clinical trials (RCTs) from the last 10 years, found in PubMed, PEDro, Scopus and Web of Science databases. Methodological quality was analyzed using the PEDro scale and the Cochrane Collaboration Risk of Bias Tool. Regarding the evaluation of functional capacity, mainly assessed were lung function, respiratory muscle strength, and maximal or aerobic exercise. The results showed that WE improves functional capacity compared to a non-exercising control group (SMD: 73.42; IC 95%: 40.40 to 106.45; I2: 0%). There are no statistically significant differences between a WE treatment and a land exercise (LE) treatment (p = 0.24) in functional capacity, nor with respect to respiratory muscle strength (p = 0.97). These data should be interpreted with caution, as more RCTs with aquatic intervention in COPD patients are needed to elucidate whether there are differences between WE or LE according to patient characteristics and comorbidities.

Adaptive Resetting of Tuberoinfundibular Dopamine (TIDA) Network Activity during Lactation in Mice.

Giving birth triggers a wide repertoire of physiological and behavioral changes in the mother to enable her to feed and care for her offspring. These changes require coordination and are often orchestrated from the CNS, through as of yet poorly understood mechanisms. A neuronal population with a central role in puerperal changes is the tuberoinfundibular dopamine (TIDA) neurons that control release of the pituitary hormone, prolactin, which triggers key maternal adaptations, including lactation and maternal care. Here, we used Ca2+ imaging on mice from both sexes and whole-cell recordings on female mouse TIDA neurons in vitro to examine whether they adapt their cellular and network activity according to reproductive state. In the high-prolactin state of lactation, TIDA neurons shift to faster membrane potential oscillations, a reconfiguration that reverses upon weaning. During the estrous cycle, however, which includes a brief, but pronounced, prolactin peak, oscillation frequency remains stable. An increase in the hyperpolarization-activated mixed cation current, Ih, possibly through unmasking as dopamine release drops during nursing, may partially explain the reconfiguration of TIDA rhythms. These findings identify a reversible plasticity in hypothalamic network activity that can serve to adapt the dam for motherhood.SIGNIFICANCE STATEMENT Motherhood requires profound behavioral and physiological adaptations to enable caring for offspring, but the underlying CNS changes are poorly understood. Here, we show that, during lactation, neuroendocrine dopamine neurons, the "TIDA" cells that control prolactin secretion, reorganize their trademark oscillations to discharge in faster frequencies. Unlike previous studies, which typically have focused on structural and transcriptional changes during pregnancy and lactation, we demonstrate a functional switch in activity and one that, distinct from previously described puerperal modifications, reverses fully on weaning. We further provide evidence that a specific conductance (Ih) contributes to the altered network rhythm. These findings identify a new facet of maternal brain plasticity at the level of membrane properties and consequent ensemble activity.

Glucosinolate-Degradation Products as Co-Adjuvant Therapy on Prostate Cancer in Vitro.

Glucosinolate-degradation products (GS-degradation products) are believed to be responsible for the anticancer effects of cruciferous vegetables. Furthermore, they could improve the efficacy and reduce side-effects of chemotherapy. The aim of the present study was to determine the cytotoxic effects of GS-degradation products on androgen-insensitive human prostate cancer (AIPC) PC-3 and DU 145 cells and investigate their ability to sensitize such cells to chemotherapeutic drug Docetaxel (DOCE). Cells were cultured under growing concentrations of allyl-isothiocyanate (AITC), sulforaphane (SFN), 4-pentenyl-isothiocyanate (4PI), iberin (IB), indole-3-carbinol (I3C), or phenethyl-isothiocyanate (PEITC) in absence or presence of DOCE. The anti-tumor effects of these compounds were analyzed using the trypan blue exclusion, apoptosis, invasion and RT-qPCR assays and confocal microscopy. We observed that AITC, SFN, IB, and/or PEITC induced a dose- and time-dependent cytotoxic effect on PC-3 and DU 145 cells, which was mediated, at least, by apoptosis and cell cycle arrest. Likewise, we showed that these GS-degradation products sensitized both cell lines to DOCE by synergic mechanisms. Taken together, our results indicate that GS-degradation products can be promising compounds as co-adjuvant therapy in prostate cancer.

PDGFB, a new candidate plasma biomarker for venous thromboembolism: results from the VEREMA affinity proteomics study.

There is a clear clinical need for high-specificity plasma biomarkers for predicting risk of venous thromboembolism (VTE), but thus far, such markers have remained elusive. Utilizing affinity reagents from the Human Protein Atlas project and multiplexed immuoassays, we extensively analyzed plasma samples from 2 individual studies to identify candidate protein markers associated with VTE risk. We screened plasma samples from 88 VTE cases and 85 matched controls, collected as part of the Swedish "Venous Thromboembolism Biomarker Study," using suspension bead arrays composed of 755 antibodies targeting 408 candidate proteins. We identified significant associations between VTE occurrence and plasma levels of human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1), von Willebrand factor (VWF), glutathione peroxidase 3 (GPX3), and platelet-derived growth factor ß (PDGFB). For replication, we profiled plasma samples of 580 cases and 589 controls from the French FARIVE study. These results confirmed the association of VWF and PDGFB with VTE after correction for multiple testing, whereas only weak trends were observed for HIVEP1 and GPX3. Although plasma levels of VWF and PDGFB correlated modestly (ρ ∼ 0.30) with each other, they were independently associated with VTE risk in a joint model in FARIVE (VWF P < .001; PDGFB P = .002). PDGFΒ was verified as the target of the capture antibody by immunocapture mass spectrometry and sandwich enzyme-linked immunosorbent assay. In conclusion, we demonstrate that high-throughput affinity plasma proteomic profiling is a valuable research strategy to identify potential candidate biomarkers for thrombosis-related disorders, and our study suggests a novel association of PDGFB plasma levels with VTE.

Immunocapture strategies in translational proteomics.

Aiming at clinical studies of human diseases, antibody-assisted assays have been applied to biomarker discovery and toward a streamlined translation from patient profiling to assays supporting personalized treatments. In recent years, integrated strategies to couple and combine antibodies with mass spectrometry-based proteomic efforts have emerged, allowing for novel possibilities in basic and clinical research. Described in this review are some of the field's current and emerging immunocapture approaches from an affinity proteomics perspective. Discussed are some of their advantages, pitfalls and opportunities for the next phase in clinical and translational proteomics.

Effects of Brassicaceae Isothiocyanates on Prostate Cancer.

Despite the major progress made in the field of cancer biology, cancer is still one of the leading causes of mortality, and prostate cancer (PCa) is one of the most encountered malignancies among men. The effective management of this disease requires developing better anticancer agents with greater efficacy and fewer side effects. Nature is a large source for the development of chemotherapeutic agents, with more than 50% of current anticancer drugs being of natural origin. Isothiocyanates (ITCs) are degradation products from glucosinolates that are present in members of the family Brassicaceae. Although they are known for a variety of therapeutic effects, including antioxidant, immunostimulatory, anti-inflammatory, antiviral and antibacterial properties, nowadays, cell line and animal studies have additionally indicated the chemopreventive action without causing toxic side effects of ITCs. In this way, they can induce cell cycle arrest, activate apoptosis pathways, increase the sensitivity of resistant PCa to available chemodrugs, modulate epigenetic changes and downregulate activated signaling pathways, resulting in the inhibition of cell proliferation, progression and invasion-metastasis. The present review summarizes the chemopreventive role of ITCs with a particular emphasis on specific molecular targets and epigenetic alterations in in vitro and in vivo cancer animal models.

Parallel barcoding of antibodies for DNA-assisted proteomics.

DNA-assisted proteomics technologies enable ultra-sensitive measurements in multiplex format using DNA-barcoded affinity reagents. Although numerous antibodies are available, nowadays targeting nearly the complete human proteome, the majority is not accessible at the quantity, concentration, or purity recommended for most bio-conjugation protocols. Here, we introduce a magnetic bead-assisted DNA-barcoding approach, applicable for several antibodies in parallel, as well as reducing required reagents quantities up to a thousand-fold. The success of DNA-barcoding and retained functionality of antibodies were demonstrated in sandwich immunoassays and standard quantitative Immuno-PCR assays. Specific DNA-barcoding of antibodies for multiplex applications was presented on suspension bead arrays with read-out on a massively parallel sequencing platform in a procedure denoted Immuno-Sequencing. Conclusively, human plasma samples were analyzed to indicate the functionality of barcoded antibodies in intended proteomics applications.

Magnetic bead assisted labeling of antibodies at nanogram scale.

There are currently several initiatives that aim to produce binding reagents for proteome-wide analysis. To enable protein detection, visualization, and target quantification, covalent coupling of reporter molecules to antibodies is essential. However, current labeling protocols recommend considerable amount of antibodies, require antibody purity and are not designed for automation. Given that small amounts of antibodies are often sufficient for downstream analysis, we developed a labeling protocol that combines purification and modification of antibodies at submicrogram quantities. With the support of magnetic microspheres, automated labeling of antibodies in parallel using biotin or fluorescent dyes was achieved.

Plasma levels of complement components C5 and C9 are associated with thrombin generation.

BACKGROUND: The thrombin generation assay (TGA) evaluates the potential of plasma to generate thrombin over time, providing a global picture of an individual's hemostatic balance. OBJECTIVES: This study aimed to identify novel biological determinants of thrombin generation using a multiomics approach. METHODS: Associations between TGA parameters and plasma levels of 377 antibodies targeting 236 candidate proteins for cardiovascular risk were tested using multiple linear regression analysis in 770 individuals with venous thrombosis from the Marseille Thrombosis Association (MARTHA) study. Proteins associated with at least 3 TGA parameters were selected for validation in an independent population of 536 healthy individuals (Etablissement Français du Sang Alpes-Méditerranée [EFS-AM]). Proteins with strongest associations in both groups underwent additional genetic analyses and in vitro experiments. RESULTS: Eighteen proteins were associated (P < 1.33 × 10⁻4) with at least 3 TGA parameters in MARTHA, among which 13 demonstrated a similar pattern of associations in EFS-AM. Complement proteins C5 and C9 had the strongest associations in both groups. Ex vivo supplementation of platelet-poor plasma with purified C9 protein had a significant dose-dependent effect on TGA parameters. No effect was observed with purified C5. Several single nucleotide polymorphisms associated with C5 and C9 plasma levels were identified, with the strongest association for the C5 missense variant rs17611, which was associated with a decrease in C5 levels, endogenous thrombin potential, and peak in MARTHA. No association of this variant with TGA parameters was observed in EFS-AM. CONCLUSION: This study identified complement proteins C5 and C9 as potential determinants of thrombin generation. Further studies are warranted to establish causality and elucidate the underlying mechanisms.

Telerehabilitation, A Viable Option in Patients with Persistent Post-COVID Syndrome: A Systematic Review.

The number of patients with post-COVID-19 syndrome continues to increase considerably, having serious healthcare, social and economic repercussions. The objective of this study is to describe the effectiveness of telerehabilitation to alleviate the symptoms of post-COVID-19 syndrome. A systematic review was conducted using the information available on four databases (PubMed, Medline, Scielo and PEDRo) on these patients until November 2022. The MeSH search terms were: Post-COVID syndrome, Post-COVID-19, Long COVID, Telerehabilitation, Physiotherapy, Rehabilitation, Virtual, Home care. Six articles were included which provided information on 140 patients, detailing their symptomatology, assessment, treatment and monitoring. The variables measured were dyspnea, fatigue, physical performance and quality of life. All studies included aerobic and anaerobic exercises. Most notable among the techniques used were rib cage expansion exercises, respiratory control and thoracic cage stretching, patient education, Mindfulness and virtual reality games to address physical, mental and relaxation aspects. The use of telerehabilitation could be an effective tool for the treatment of persistent symptoms after suffering from COVID-19. It has been shown in these studies that patients improve both their physical performance and their quality of life.

Biliverdin Reductase B Is a Plasma Biomarker for Intraplaque Hemorrhage and a Predictor of Ischemic Stroke in Patients with Symptomatic Carotid Atherosclerosis.

BACKGROUND: Intraplaque hemorrhage (IPH) is a hallmark of atherosclerotic plaque instability. Biliverdin reductase B (BLVRB) is enriched in plasma and plaques from patients with symptomatic carotid atherosclerosis and functionally associated with IPH. OBJECTIVE: We explored the biomarker potential of plasma BLVRB through (1) its correlation with IPH in carotid plaques assessed by magnetic resonance imaging (MRI), and with recurrent ischemic stroke, and (2) its use for monitoring pharmacotherapy targeting IPH in a preclinical setting. METHODS: Plasma BLVRB levels were measured in patients with symptomatic carotid atherosclerosis from the PARISK study (n = 177, 5 year follow-up) with and without IPH as indicated by MRI. Plasma BLVRB levels were also measured in a mouse vein graft model of IPH at baseline and following antiangiogenic therapy targeting vascular endothelial growth factor receptor 2 (VEGFR-2). RESULTS: Plasma BLVRB levels were significantly higher in patients with IPH (737.32 ± 693.21 vs. 520.94 ± 499.43 mean fluorescent intensity (MFI), p = 0.033), but had no association with baseline clinical and biological parameters. Plasma BLVRB levels were also significantly higher in patients who developed recurrent ischemic stroke (1099.34 ± 928.49 vs. 582.07 ± 545.34 MFI, HR = 1.600, CI [1.092-2.344]; p = 0.016). Plasma BLVRB levels were significantly reduced following prevention of IPH by anti-VEGFR-2 therapy in mouse vein grafts (1189 ± 258.73 vs. 1752 ± 366.84 MFI; p = 0.004). CONCLUSIONS: Plasma BLVRB was associated with IPH and increased risk of recurrent ischemic stroke in patients with symptomatic low- to moderate-grade carotid stenosis, indicating the capacity to monitor the efficacy of IPH-preventive pharmacotherapy in an animal model. Together, these results suggest the utility of plasma BLVRB as a biomarker for atherosclerotic plaque instability.

Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism.

Venous thromboembolism (VTE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. Here we show, using proteomics profiling to screen plasma from patients with suspected acute VTE, and several case-control studies for VTE, how Complement Factor H Related 5 protein (CFHR5), a regulator of the alternative pathway of complement activation, is a VTE-associated plasma biomarker. In plasma, higher CFHR5 levels are associated with increased thrombin generation potential and recombinant CFHR5 enhanced platelet activation in vitro. GWAS analysis of ~52,000 participants identifies six loci associated with CFHR5 plasma levels, but Mendelian randomization do not demonstrate causality between CFHR5 and VTE. Our results indicate an important role for the regulation of the alternative pathway of complement activation in VTE and that CFHR5 represents a potential diagnostic and/or risk predictive plasma biomarker.

Imaging and multi-omics datasets converge to define different neural progenitor origins for ATRT-SHH subgroups.

Atypical teratoid rhabdoid tumors (ATRT) are divided into MYC, TYR and SHH subgroups, suggesting diverse lineages of origin. Here, we investigate the imaging of human ATRT at diagnosis and the precise anatomic origin of brain tumors in the Rosa26-CreERT2::Smarcb1flox/flox model. This cross-species analysis points to an extra-cerebral origin for MYC tumors. Additionally, we clearly distinguish SHH ATRT emerging from the cerebellar anterior lobe (CAL) from those emerging from the basal ganglia (BG) and intra-ventricular (IV) regions. Molecular characteristics point to the midbrain-hindbrain boundary as the origin of CAL SHH ATRT, and to the ganglionic eminence as the origin of BG/IV SHH ATRT. Single-cell RNA sequencing on SHH ATRT supports these hypotheses. Trajectory analyses suggest that SMARCB1 loss induces a de-differentiation process mediated by repressors of the neuronal program such as REST, ID and the NOTCH pathway.

Proteomics in thrombosis research.

A State of the Art lecture titled "Proteomics in Thrombosis Research" was presented at the ISTH Congress in 2021. In clinical practice, there is a need for improved plasma biomarker-based tools for diagnosis and risk prediction of venous thromboembolism (VTE). Analysis of blood, to identify plasma proteins with potential utility for such tools, could enable an individualized approach to treatment and prevention. Technological advances to study the plasma proteome on a large scale allows broad screening for the identification of novel plasma biomarkers, both by targeted and nontargeted proteomics methods. However, assay limitations need to be considered when interpreting results, with orthogonal validation required before conclusions are drawn. Here, we review and provide perspectives on the application of affinity- and mass spectrometry-based methods for the identification and analysis of plasma protein biomarkers, with potential application in the field of VTE. We also provide a future perspective on discovery strategies and emerging technologies for targeted proteomics in thrombosis research. Finally, we summarize relevant new data on this topic, presented during the 2021 ISTH Congress.

Evaluation of Celiac Disease by Minimally Invasive Biomarkers in a Spanish Pediatric Population.

BACKGROUND: The diagnosis of celiac disease (CD) has been substantially improved with the availability of highly sensitive CD-specific IgA-TG2, Ig-GDP, and IgA-EMA. The European Society for Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) published (2012) and updated (2020) diagnostic criteria for CD in order to simplify CD diagnosis and to avoid biopsies in selected patients. METHODS: A prospective study including 5641 pediatric patients (0-16 years old) from January 2012 to January 2019 was performed. CD diagnosis was made according to the ESPGHAN algorithm. The objective of this study was to evaluate the utility of biomarkers and the relationship between TGA-IgA and EMA titers. RESULTS: CD diagnoses were confirmed in 113 patients, 110 were IgA-TG2-positive and 3 (2.7%) had IgA deficiency. The diagnosis was made by serologic tests in 95 (84.1%) patients. Only 18 (15.9%) patients underwent intestinal biopsy. We obtained 100% concordance between IgA-EMA and positive results for IgA-TG2 ≥ 10 ULN with IgA-EMA antibody titer ≥ 1:80. CONCLUSIONS: This study provides evidence of a positive correlation between IgA-TG2 antibody serum levels and IgA-EMA. The diagnosis could be guaranteed with strict application of IgA-TG2 values ≥ 10 ULN (confirmed by subsequent testing) plus the serological response to the gluten-free diet (GFD).

Effectiveness of positive expiratory pressure on patients over 16 years of age with cystic fibrosis: systematic review and meta-analysis.

INTRODUCTION: Cystic fibrosis (CF) is an autosomal recessive disease that involves the cells that produce mucus and sweat, affecting many organs, especially the lungs. Positive expiratory pressure (PEP) devices generate a pressure opposite to that exerted by the airways during expiration, thus improving mucociliary clearance. OBJECTIVES: To evaluate the efficacy of PEP devices as a resource to facilitate the mucus removal and other outcomes in people with CF, as well as the possible adverse effects derived from their use. MATERIAL AND METHOD: A systematic review and meta-analysis was conducted according to PRISMA standards. The descriptors were 'cystic fibrosis', 'PEP', and 'physiotherapy and/or physical therapy'. The search was performed in four databases: PubMed, PEDro, and Web of Science and Scopus, in July 2021. The inclusion criteria were randomized controlled trials (RCTs) over the last 10 years. The methodological quality of the studies was analyzed and meta-analysis was performed with Review Manager software. RESULTS: Ten RCTs met the objectives and criteria, with a total of 274 participants. The trials score a moderate methodological quality on the PEDro scale. No clear results were obtained on whether PEP provides better lung function than other breathing techniques (such as airway clearance); but it does achieve a higher rate of lung clearance than physical exercise. CONCLUSIONS: PEP is more effective than usual care or no intervention, although there is not enough evidence to confirm that PEP achieves improvements in forced expiratory volume in the first second (FEV1) compared with other techniques. It is a safe technique, without adverse effects.

Red Cell Distribution Width as a Predictive Factor of Celiac Disease in Middle and Late Adulthood and Its Potential Utility as Celiac Disease Screening Criterion.

Red cell distribution width (RDW) could be of interest by its potential use in the assessment of celiac disorder (CD). The main objective of this study was to evaluate the case positive rate of CD and the utility of red cell distribution width (RDW) in the CD diagnosis. This prospective study included 9.066 middle adult (≥45 years old) and elderly patients (≥60 years old) from 2012 to 2021. CD diagnosis was performed by CD antibody tests (serology and Human Leucocyte Antigen genotype (HLA)) and biopsy. Gastrointestinal and extra-intestinal manifestations as well as hematological and biochemical parameters were analyzed. CD diagnoses were confirmed in 101 patients (median (IQR) age = 62 (52.3−73); 68.32% women) by serologic tests (100%) and intestinal biopsy (88.12%), showing mainly marked or complete atrophy (76.24%, MARSH 3a−c). Anemia was the most commonly presenting extra-intestinal manifestation (28.57%). Among 8975 individuals without CD, 168 age and sex matched were included. By comparison of CD and no CD individuals, we observed that high >14.3% RDW was exhibited by 58.40% and 35.2% individuals with CD and without CD, respectively. Furthermore, high RDW is associated with CD and grade III atrophy. We suggest that RDW could be used as a CD screening criterion.

DGAT1 participates in the effect of HNF4A on hepatic secretion of triglyceride-rich lipoproteins.

OBJECTIVE: Hepatocyte nuclear factor-4alpha (HNF4A) is a transcription factor that influences plasma triglyceride metabolism via an as of yet unknown mechanism. In this study, we searched for the critical protein that mediates this effect using different human model systems. METHODS AND RESULTS: Up- and downregulation of HNF4A in human hepatoma Huh7 and HepG2 cells was associated with marked changes in the secretion of triglyceride-rich lipoproteins (TRLs). Short interfering RNA (siRNA) inhibition of HNF4A influenced the expression of several genes, including acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1). siRNA knockdown of DGAT1 reduced DGAT1 activity and decreased the secretion of TRLs. No additive effects of combined siRNA inhibition of HNF4A and DGAT1 were found on the secretion of TRLs, whereas the increase in TRL secretion induced by HNF4A overexpression was largely abolished by DGAT1 siRNA inhibition. A putative binding site for HNF4A was defined by in silico and in vitro methods. HNF4A and DGAT1 expressions were analyzed in 80 human liver samples, and significant relationships were observed between HNF4A and DGAT1 mRNA levels (r(2)=0.50, P<0.0001) and between DGAT1 mRNA levels and plasma triglyceride concentration (r(2)=0.09, P<0.01). CONCLUSION: This study identified DGAT1 as an important protein that participates in the effect of HNF4A on hepatic secretion of TRLs.

Affinity Proteomics Assays for Cardiovascular and Atherosclerotic Disease Biomarkers.

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.

Bead-Based Assays for Validating Proteomic Profiles in Body Fluids.

Protein biomarkers in biological fluids represent an important resource for improving the clinical management of diseases. Current proteomics technologies are capable of performing high-throughput and multiplex profiling in different types of fluids, often leading to the shortlisting of tens of candidate biomarkers per study. However, before reaching any clinical setting, these discoveries require thorough validation and an assay that would be suitable for routine analyses. In the path from biomarker discovery to validation, the performance of the assay implemented for the intended protein quantification is extremely critical toward achieving reliable and reproducible results. Development of robust sandwich immunoassays for individual candidates is challenging and labor and resource intensive, and multiplies when evaluating a panel of interesting candidates at the same time. Here we describe a versatile pipeline that facilitates the systematic and parallel development of multiple sandwich immunoassays using a bead-based technology.