Multiplex Methodology Research Articles

Multiplex, high-throughput method to study cancer and immune cell mechanotransduction

Studying cellular mechanoresponses during cancer metastasis is limited by sample variation or complex protocols that current techniques require. Metastasis is governed by mechanotransduction, whereby cells translate external stimuli, such as circulatory fluid shear stress (FSS), into biochemical cues. We present high-throughput, semi-automated methods to expose cells to FSS using the VIAFLO96 multichannel pipetting device custom-fitted with 22 G needles, increasing the maximum FSS 94-fold from the unmodified tips. Specifically, we develop protocols to semi-automatically stain live samples and to fix, permeabilize, and intracellularly process cells for flow cytometry analysis. Our first model system confirmed that the pro-apoptotic effects of TRAIL therapeutics in prostate cancer cells can be enhanced via FSS-induced Piezo1 activation. Our second system implements this multiplex methodology to show that FSS exposure (290 dyn cm−2) increases activation of murine bone marrow-derived dendritic cells. These methodologies greatly improve the mechanobiology workflow, offering a high-throughput, multiplex approach.

UNLEASHING SOUTH CHINA SEA CONFLICT: MULTIPLEX ANALYSIS OF DIPLOMATIC CHAOS

The South China Sea, one of the busiest trade routes around the world consisting of an area of 3.5 million square kilometers, is subject to several overlapping territorial disputes involving China, Vietnam, the Philippines, Taiwan, Malaysia, and Brunei as its stakeholders. The South China Sea connects Asia to Europe and Africa. Political rivals have tried amicable settlement of the contemporary disputes but instead, the tensions have increased over the course of recent years. Multiple Confidence Building Measures (CBMs) and Treaties of Amity and Cooperation (TAC) have been signed by the contending states but all have proven to be in vain. Mechanisms like Freedom of Navigation Operations (FONOPs) are used for chest thumping and hard power display. Numerous other factors are there that eventuate in weakening of diplomatic ties between the contending states. One of such factors is involved is the presence of alien powers like the United States for maintenance of a World Order favouring her on interests under the guise of providing security to the region. Adopting multiplex methodology, this research paper adopts an approach aiming at giving a synopsis of the inherent disputes between the stakeholders, the reasons for failure of efforts for amicable settlement, the role of the stakeholders with special reference to the major contending forces i.e., United States and China, the subsequent implications arising therefrom and some possible solutions to the issue. Keywords: SCS, FONOS, CBM, Multiplex methodology.

Design and analysis of a fiber-optic sensing system for shape reconstruction of a minimally invasive surgical needle

This paper presents the performance analysis of the system for real-time reconstruction of the shape of the rigid medical needle used for minimally invasive surgeries. The system is based on four optical fibers glued along the needle at 90 degrees from each other to measure distributed strain along the needle from four different sides. The distributed measurement is achieved by the interrogator which detects the light scattered from each section of the fiber connected to it and calculates the strain exposed to the fiber from the spectral shift of that backscattered light. This working principle has a limitation of discriminating only a single fiber because of the overlap of backscattering light from several fibers. In order to use four sensing fibers, the Scattering-Level Multiplexing (SLMux) methodology is applied. SLMux is based on fibers with different scattering levels: standard single-mode fibers (SMF) and MgO-nanoparticles doped fibers with a 35–40 dB higher scattering power. Doped fibers are used as sensing fibers and SMFs are used to spatially separate one sensing fiber from another by selecting appropriate lengths of SMFs. The system with four fibers allows obtaining two pairs of opposite fibers used to reconstruct the needle shape along two perpendicular axes. The performance analysis is conducted by moving the needle tip from 0 to 1 cm by 0.1 cm to four main directions (corresponding to the locations of fibers) and to four intermediate directions (between neighboring fibers). The system accuracy for small bending (0.1–0.5 cm) is 90% and for large bending (0.6–1 cm) is approximately 92%.

Mitigation of Phase Noise and Nonlinearities for High Capacity Radio-over-Fiber Links

Radio-over-fiber (RoF) links successfully provide high data rates and bandwidth capacity with a low complexity system architecture, as compared to its counterpart digital-RoF. In addition, the compound of quadrature amplitude modulation (QAM) and orthogonal frequency division multiplexed (OFDM) modulation schemes further enhance the process of these achievements. However, high data rates and bandwidth-capacity-supported RoF links face nonlinearities (NLs), linear distortions (LDs), and phase noise challenges that degrade the reliability of communication networks (CNs). Therefore, in this paper, to suppress NLs, LDs, and phase noise, next generation cloud radio access networks (CRANs) are investigated using RoF links and wavelength division multiplexing (WDM) methodology based on 16, 32, and 64 QAM-OFDM modulation schemes. The receiver of the proposed framework is designed, applying an improved digital signal processing (DSP) system that includes overlap frequency domain equalization (OFDE), a synchronization process, and time domain equalization (TDE). Theoretical and simulation models are organized for estimating the proposed RoF link with the aid of different values of transmission ranges, input power, output power, bit rate, bits per symbol, channel spacing, and the number of users. The fitness of the model matches that of existing approaches.

Peacebuilding through the Lens of an Emancipatory Peacebuilding Paradigm

Critical and Emancipatory Theory (CET) of peacebuilding emerged as the sixth school of thought in Peace and Conflict Studies to critique the liberal and neoliberal approaches to peacebuilding. CET contends that liberal and neoliberal approaches to peacebuilding are discriminatory and biased, perpetrates the interest of Western elites, hinders the achievement of social justice, and considers the local as insignificant for peacebuilding. A call for the reformation of the liberal and neoliberal approaches necessitated the CET school of thought to outline certain principles and guidelines that could guide the practice of peace-building. Different methodologies, intervention strategies, principles and approaches that could guide praxis have therefore been advanced by scholars, researchers, and practitioners. This paper therefore examines and reflects on the multiplex methodologies in peacebuilding, the adoption of a multidisciplinary approach to understanding peace and peacebuilding, and the question of intervention in post-war peacebuilding, the principles that guide the implementation of peacebuilding from the lens of Critical and Emancipatory school of thought. This piece contend that CET approach may not be self-sufficient but would be the most appropriate way to decentralize the peacebuilding process and, as such, local or indigenous peacebuilding processes must be encouraged while acknowledging the salient role of the international community as well.

Successful elevation of circulating acetate and propionate by dietary modulation does not alter T-regulatory cell or cytokine profiles in healthy humans: a pilot study.

Increased circulating concentrations of short-chain fatty acids (SCFA) achieved by ingestion of high-fibre diets is associated with anti-inflammatory effects through promotion of FoxP3+ regulatory T(reg) cells in mouse models. This study aimed to determine whether similar increments in blood SCFA levels can be achieved in humans and whether these are associated with similar immune modulatory effects. In a pilot single-blinded, randomised, controlled cross-over study in ten healthy subjects, the effects were determined of high- (39g/day) and low-fibre (18g/day) intake (all food provided) on SCFA (gas chromatography), proportions of Treg cells (flow cytometry) and a panel of cytokines (multiplex methodology) measured in peripheral blood at day 5 of each diet. Actual fibre intake differed between the diets by 19 [16-21] g/day (P< 0.001). Median [range] total plasma SCFA levels with high-fibre intake were 174.5 [104.8-249.5] µmol/L, which were greater than those associated with low-fibre intake at 59.0 [26.5-79.9] (P < 0.001). Differences were significantly different for both acetate and propionate. The frequencies of total CD4 T cells and T-regulatory cells, and concentrations of inflammatory and anti-inflammatory cytokines were not significantly different between the dietary interventions. Plasma SCFA levels can be modulated by altering dietary fibre consumption in healthy individuals with increments similar to those achieved in murine studies. Five days of diet intervention did not result in changes in regulatory T-cell proportions and cytokine concentrations in peripheral blood, and may require longer duration of dietary change.

Sequentially multiplexed amperometry for electrochemical biosensors

Multiplexed electrochemical biosensors are intriguing due to their capability to permit high-throughput and low-cost assays. While commercial single-chip potentiostats are one promising approach for rapidly prototyping portable and low-cost electrochemical biosensors, it is still challenging to utilize them to achieve parallel multiplexing due to the limited resources integrated onto the chips. In this paper, we provide a methodology for incorporating multiplexing into commercial single-chip potentiostats by using a sequential architecture. In the sequential architecture, the multiplexed biosensors are interfaced to the single-chip potentiostat via single-pole single-throw switches, and the measurements alternate across the sensors. We build analytical and finite element models to investigate the behavior of the sensors, particularly when they are disconnected from the potentiostat, and find that we can take advantage of the dynamics of the sensors to achieve improved sensitivity over conventional chronoamperometry. We also investigate and compare different strategies to interface the multiplexed sensors to the single-chip potentiostat. Using the proposed multiplexing architecture, we demonstrate the implementation of 16-fold multiplexed amperometry, which is validated using ferricyanide measurement. Finally, the sequential multiplexing methodology is applied to a multiplexed bead-based electronic enzyme-linked immunosorbent assays of human interleukin-6.

QMC-PCRx: a novel method for rapid mutation detection

AimsWe previously described the quick multiplex consensus PCR (QMC-PCR) as a method for rapid mutation screening in low-quality template. QMC-PCR has two-stages: a prediagnostic multiplex (PDM) reaction followed by a...

Fabry Disease Biomarkers: Analysis of Urinary Lyso-Gb3 and Seven Related Analogs Using Tandem Mass Spectrometry.

Fabry disease is an X-linked lysosomal storage disorder caused by the absence or reduction of the enzyme α-galactosidase A activity. Currently, globotriaosylsphingosine (lyso-Gb3 ) and globotriaosylceramide (Gb3 ) are used as biomarkers to diagnose and monitor Fabry patients. However, recent metabolomic studies have shown that several glycosphingolipids are also elevated in biological fluids of affected patients and may be related to disease manifestations. This unit describes a multiplex methodology targeting the analysis of urinary lyso-Gb3 and seven structurally related analogs. A solid-phase extraction process is performed, then lyso-Gb3 and its analogs are analyzed simultaneously with an internal standard by ultra-performance liquid chromatography (UPLC) coupled to a tandem mass spectrometry (MS/MS) system. This methodology can be useful for the diagnosis of Fabry patients, including patients with cardiac variant mutations, but also to monitor the efficacy of therapeutic interventions, considering that lyso-Gb3 analogs are more elevated than lyso-Gb3 itself in urine. © 2016 by John Wiley & Sons, Inc.

Complete photoionization experiments via ultrafast coherent control with polarization multiplexing. II. Numerics and analysis methodologies

The feasibility of complete photoionization experiments, in which the full set of photoionization matrix elements are determined, using multiphoton ionization schemes with polarization-shaped pulses has recently been demonstrated [Hockett et. al., Phys. Rev. Lett. 112, 223001 (2014)]. Here we extend on our previous work to discuss further details of the numerics and analysis methodology utilised, and compare the results directly to new tomographic photoelectron measurements, which provide a more sensitive test of the validity of the results. In so doing we discuss in detail the physics of the photoionziation process, and suggest various avenues and prospects for this coherent multiplexing methodology.

Data mining strategies to improve multiplex microbead immunoassay tolerance in a mouse model of infectious diseases.

Multiplex methodologies, especially those with high-throughput capabilities generate large volumes of data. Accumulation of such data (e.g., genomics, proteomics, metabolomics etc.) is fast becoming more common and thus requires the development and implementation of effective data mining strategies designed for biological and clinical applications. Multiplex microbead immunoassay (MMIA), on xMAP or MagPix platform (Luminex), which is amenable to automation, offers a major advantage over conventional methods such as Western blot or ELISA, for increasing the efficiencies in serodiagnosis of infectious diseases. MMIA allows detection of antibodies and/or antigens efficiently for a wide range of infectious agents simultaneously in host blood samples, in one reaction vessel. In the process, MMIA generates large volumes of data. In this report we demonstrate the application of data mining tools on how the inherent large volume data can improve the assay tolerance (measured in terms of sensitivity and specificity) by analysis of experimental data accumulated over a span of two years. The combination of prior knowledge with machine learning tools provides an efficient approach to improve the diagnostic power of the assay in a continuous basis. Furthermore, this study provides an in-depth knowledge base to study pathological trends of infectious agents in mouse colonies on a multivariate scale. Data mining techniques using serodetection of infections in mice, developed in this study, can be used as a general model for more complex applications in epidemiology and clinical translational research.

Architecture of a modular, multichannel readout system for dense electrochemical biosensor microarrays

The architecture of a modular, multichannel readout system for dense electrochemical microarrays, targeting Lab-on-a-Chip applications, is presented. This approach promotes efficient component reusability through a hybrid multiplexing methodology, maintaining high levels of sampling performance and accuracy. Two readout modes are offered, which can be dynamically interchanged following signal profiling, to cater for both rapid signal transitions and weak current responses. Additionally, functional extensions to the described architecture are discussed, which provide the system with multi-biasing capabilities. A prototype integrated circuit of the proposed architecture’s analog core and a supporting board were implemented to verify the working principles. The system was evaluated using standard loads, as well as electrochemical sensor arrays. Through a range of operating conditions and loads, the prototype exhibited a highly linear response and accurately delivered the readout of input signals with fast transitions and wide dynamic ranges.

Maternal inflammatory markers and term labor performance

We sought to examine the relationship between maternal markers of inflammation and labor performance. A nested cohort study was performed utilizing an established cohort of term nulliparous patients. Maternal blood was collected at the onset of regular, painful contractions in patients undergoing labor induction or at admission in patients with spontaneous labor. Levels of cytokines including interleukin (IL)-1, IL-6, and tumor necrosis factor-α were determined using standard multiplex methodology. Maternal demographic data were collected prospectively. Detailed retrospective chart review was performed to extract data on cervical dilation, effacement, and station during labor. Subjects were excluded if they failed to achieve complete dilation. Mixed effects modeling was used to examine the association between serum cytokine quartiles and labor progress in the latent and active phases. In all, 334 women were included in our analysis. The lowest quartile of IL-6 was associated with slower latent labor (P = .001). In contrast, the highest quartiles of IL-1 and tumor necrosis factor-α were associated with slower active labor (P = .03 and .0002, respectively). Proinflammatory activation is important in labor initiation. However, once active labor is established, excess inflammation can be detrimental to efficient labor progress. These data may explain, in part, the known associations among clinical chorioamnionitis, cesarean delivery, and postpartum hemorrhage.

Multiplex Analysis of Novel Urinary Lyso-Gb3-Related Biomarkers for Fabry Disease by Tandem Mass Spectrometry

Fabry disease is a lysosomal storage disorder caused by the absence or reduction of α-galactosidase A enzyme activity. The enzymatic deficiency results in the impaired catabolism of neutral sphingolipids with terminal α-galactosyl residues and subsequent accumulation in several tissues. Biomarkers reflecting disease severity and progression, the response to therapeutic intervention, and details of molecular pathogenesis are needed. Until now, two sphingolipids were targeted as biomarkers in urine and plasma of Fabry patients: globotriaosylceramide (Gb(3)) and globotriaosylsphingosine (lyso-Gb(3)). Using metabolomic approaches, our group recently discovered seven novel urinary lyso-Gb(3)-related Fabry disease biomarkers with mass-to-charge ratios (m/z) of 758, 774, 784, 800, 802, 820, and 836. All these biomarkers exhibited modifications of the lyso-Gb(3) sphingosine moiety. The aims of the present study were to devise and validate a specific tandem mass spectrometry multiplex methodology for the relative quantification of these seven analogues and to evaluate their urinary excretion levels in samples from 164 Fabry patients and 94 healthy controls. We found no detectable analogues in healthy controls, except for trace amounts of the analogue with m/z 836. Significant correlations were established between lyso-Gb(3) analogue levels in urine and gender (p < 0.001). Fabry males had higher excretion levels compared to females with the disease. Lyso-Gb(3) analogue levels correlated well with enzyme replacement therapy (ERT) status in males (p < 0.05). The urinary analogue distributions varied among Fabry patients. However, the analogues with m/z 802, 820, and 836 were generally more abundant in the majority of patients. Lyso-Gb(3) analogues are promising urinary biomarkers for Fabry disease.

Biomarkers in Inflammatory Childhood Diseases

Biomarkers are of crucial importance in modern medicine for risk-assessment, disease prevention, early diagnosis, drug target identification, drug response and monitoring of disease activity. Biomarkers can be defined as characteristics which are measured and evaluated as indicators of a normal biologic, or pathologic process, or pharmacologic responses towards a therapeutic intervention [1]. Although the term biomarker is relatively new, biomarkers have been used for considerable time, in fact as long as clinical medicine. For example, body temperature is a well-known biomarker for fever; serum levels of C-reactive protein (the most performed assay in hospital laboratories) is a marker for inflammation and glucose levels are used for diagnosis as well as therapeutic readout for controlling diabetes. For this special issue on Biomarkers in Inflammatory Childhood Diseases we took particular interest in studies dealing with biomarkers for immune mediated childhood diseases. Besides the fact that biomarkers for childhood diseases are, due to ethical restrictions, small population sizes, and limited sample volumes less available, in most cases the normal reference range is extrapolated from adult populations or animal models and therefore not always applicable for paediatric patients. As the pediatric population is in the process of development, both mentally and physically they are at risk for other hazards in comparison with adults. Furthermore, when studying the various parameters of the human immune system changes are observed during aging which have to be taken in account in paediatric patients [2]. Therefore, insight and progression of complex paediatric diseases, in particular inflammatory diseases, can benefit from establishment and validation of a comprehensive set of biomarkers. In this special Issue on Biomarkers in Inflammatory Childhood Diseases, various paediatric diseases are discussed which are either chronic such as cystic fibrosis and allergic rhinitis or asthma, or acute disease with life threatening complications such as paediatric pulmonary hypertension and H1N1 infection. Of interest to note is that there are several contributions which use or discuss non invasive sample to acquire biological material of a diseased organ, such as exhaled breath condensate from asthmatic children, and faeces of cystic fibrosis patients with potential intestinal inflammation. Furthermore, for optimal data mining several researchers also implemented state of the art new multiplex technologies which can handle micro volumes of biological samples, and produce more insight information in comparison with classical methodologies. Duncan and colleagues from Colorado, USA, report promising new data on circulating cytokines, in particular EGF, IL-6 and VEGF, which could predict adverse events in children with pulmonary hypertension. In a small cohort of children with H1N1 infection, Chiaretti et al. from Rome, Italy, show that elevated IL-1β and IL-6 are associated with disease severity, but not with ultimate outcome. Moed et al. have investigated the value of a set of both cellular as well as cytokine biomarkers in children with allergic rhinitis. While the biomarker profile was of use in establishing the severity of the allergic rhinitis, long term follow-up demonstrated reduction of allergic symptoms with persistence of the in vitro cellular abnormalities. Also in predicting which children with wheezing symptoms would progress to asthma, as was studied by Klaassen et al. in Maastricht, clinical symptoms are better predictors than biomarkers in exhaled breath condensate. In cystic fibrosis, pulmonary inflammation is a major clinical expression of the disease and in the majority of cases respiratory failure is the cause of mortality. Accumulating data now show that intestinal inflammation contributes to the disease and Lee et al. from Sidney, Australia review this evidence, in particular the value of faecal inflammatory markers such as calprotectin and S100A12. The final paper deals with childhood obesity. Adipose tissue is a major source for adipokines, which include hormones such as leptin and adiponectin, cytokines, as well as proteins like apelin and resistin. Machura et al. from Zabrze in Poland have evaluated these adipokines in children with atopic dermatitis and find apelin and visfatin excellent biomarkers for atopic dermatitis. This special issue on Biomarkers in Inflammatory Childhood Diseases thus reflects the diversity of research in this field and shows the potential of biomarker research in paediatrics. Taken together, the strength of combining non-invasive sampling of biological samples such as saliva, urine, faeces and exhaled breath condensates, as well as multiplex analysis methodologies which can handle small sized sample volumes holds the future for biomarker research in paediatric populations. Wilco de Jager Dirk Holzinger Ger T. Rijkers

Systemic biomarkers of inflammation and haemostasis in patients with chronic necrotizing pulmonary aspergillosis

BackgroundThe purpose of this study was to investigate mediators of inflammation and haemostasis in patients with chronic necrotizing pulmonary aspergillosis (CNPA), a locally, destructive process of the lung due to invasion by Aspergillus species.MethodsMeasurements of selected biomarkers in 10 patients with CNPA and 19 healthy, matched controls were performed with enzyme-linked immunosorbent assay (ELISA) and multiplex methodology. The gene expressions of relevant biomarkers were analyzed with real-time quantitative RT-PCR.ResultsIncreased concentrations of circulating mediators of inflammation interleukin (IL)-6, IL-8, RANTES, TNF-α, ICAM-1 and mediators involved in endothelial activation and thrombosis (vWF, TF and PAI-1) were observed in patients with CNPA. The concentration of the anti-inflammatory cytokine IL-10 was increased both in plasma and in PBMC in the patient population. The gene expression of CD40L was decreased in PBMC from the patient group, accompanied by decreased concentrations of soluble (s) CD40L in the circulation.ConclusionsThe proinflammatory response against Aspergillus may be counteracted by reduced CD40L and sCD40L, as well as increased IL-10, which may compromise the immune response against Aspergillus in patients with CNPA.

Simultaneous analysis of cerebrospinal fluid biomarkers using microsphere-based xMAP multiplex technology for early detection of Alzheimer’s disease

The xMAP-Luminex multiplex platform for measurement of Alzheimer’s disease (AD) cerebrospinal fluid (CSF) biomarkers using Innogenetics AlzBio3 immunoassay reagents that are for research use only has been shown to be an effective tool for early detection of an AD-like biomarker signature based on concentrations of CSF Aβ1–42, t-tau and p-tau181. Among the several advantages of the xMAP-Luminex platform for AD CSF biomarkers are: a wide dynamic range of ready-to-use calibrators, time savings for the simultaneous analyses of three biomarkers in one analytical run, reduction of human error, potential of reduced cost of reagents, and a modest reduction of sample volume as compared to conventional enzyme-linked immunosorbant assay (ELISA) methodology. Recent clinical studies support the use of CSF Aβ1–42, t-tau and p-tau181 measurement using the xMAP-Luminex platform for the early detection of AD pathology in cognitively normal individuals, and for prediction of progression to AD dementia in subjects with mild cognitive impairment (MCI). Studies that have shown the prediction of risk for progression to AD dementia by MCI patients provide the basis for the use of CSF Aβ1–42, t-tau and p-tau181 testing to assign risk for progression in patients enrolled in therapeutic trials. Furthermore emerging study data suggest that these pathologic changes occur in cognitively normal subjects 20 or more years before the onset of clinically detectable memory changes thus providing an objective measurement for use in the assessment of treatment effects in primary treatment trials. However, numerous previous ELISA and Luminex-based multiplex studies reported a wide range of absolute values of CSF Aβ1–42, t-tau and p-tau181 indicative of substantial inter-laboratory variability as well as varying degrees of intra-laboratory imprecision. In order to address these issues a recent inter-laboratory investigation that included a common set of CSF pool aliquots from controls as well as AD patients over a range of normal and pathological Aβ1–42, t-tau and p-tau181 values as well as agreed-on standard operating procedures (SOPs) assessed the reproducibility of the multiplex methodology and Innogenetics AlzBio3 immunoassay reagents. This study showed within-center precision values of 5% to a little more than 10% and good inter-laboratory %CV values (10–20%). There are several likely factors influencing the variability of CSF Aβ1–42, t-tau and p-tau181 measurements. In this review, we describe the pre-analytical, analytical and post-analytical sources of variability including sources inherent to kits, and describe procedures to decrease the variability. A CSF AD biomarker Quality Control program has been established and funded by the Alzheimer Association, and global efforts are underway to further define optimal pre-analytical SOPs and best practices for the methodologies available or in development including plans for production of a standard reference material that could provide for a common standard against which manufacturers of immunoassay kits would assign calibration standard values.

Simultaneous Detection of Infectious Human Echoviruses and Adenoviruses by an In Situ Nuclease-Resistant Molecular Beacon-Based Assay

A multiplex methodology using two nuclease-resistant molecular beacons that target specific genomic regions of adenovirus 2 and echovirus 17 during simultaneous infection in A549 cells is presented. Using fluorescence microscopy, visualization of enteroviral and adenoviral replication was possible within 3 h postinfection.

Simultaneous measurement of multiple ear proteins with multiplex ELISA assays

A recent advancement in enzyme-linked immunosorbent assay (ELISA) technology is the multiplex antibody array that measures multiple proteins simultaneously within a single sample. This allows reduction in sample volume, time, labor, and material costs, while increasing sensitivity over single ELISA. Current multiplex platforms include planar-based systems using microplates or slides, or bead-based suspension assay with microspheres. To determine the applicability of this technology for ear research, we used 3 different multiplex ELISA-based immunoassay arrays from 4 different companies to measure cytokine levels in mouse middle and inner ear tissue lysate extracts 24 h following transtympanic Haemophilus influenzae inoculation. Middle and inner ear tissue lysates were analyzed using testing services from Quansys Biosciences, Aushon Biosystems SearchLight (both microplate-based), MILLIPLEX MAP Sample (bead-based), and a RayBiotech, Inc (slide-based) kit. Samples were assayed in duplicate or triplicate. Results were compared to determine their relative sensitivity and reliability for measures of cytokines related to inflammation. The cytokine pg/ml amounts varied among the multiplex assays, so a comparison also was made of the mean fold increase in cytokines from untreated controls. Several cytokines and chemokines were elevated, the extent dependent upon the assay sensitivity. Those most significantly elevated were IL-1α, IL-1β, IL-6, TNFα, VEGF, and IL-8/MIP-2. The results of the multiplex systems were compared with single ELISA kits (IL-1β, IL-6) to assess sensitivity over the traditional method. Overall, the Quansys Biosciences and SearchLight arrays showed the greatest sensitivity, both employing the same multiplex methodology of a spotted array within a microplate well with chemiluminescent detection. They also were more sensitive than the traditional single ELISA performed with commercial kits and matched gene expression changes determined by quantitative RT-PCR. The Quansys array showed a limit of detection for ear IL-6 down to 2–4 pg/ml, indicating it is sufficiently sensitive to detect ear proteins present in low concentrations. Thus, the multiplex ELISA procedures appear suitable and reliable for the study of hearing related proteins, providing accurate, quantitative, reproducible results with considerable improvement in sensitivity and economy.

Validation of multiplex microbead immunoassay for simultaneous serodetection of multiple infectious agents in laboratory mouse

Multiplex methodologies enable simultaneous detection of antibodies against several infectious agents allowing sample conservation, cost effectiveness, and amenability to high-throughput/automation. We have previously described a multiplex microbead immunoassay for serodetection of ten, high-priority mouse infectious pathogens. Here, we present a validation of this multiplex diagnostic system using approximately four hundred serum samples from different groups of mice. Computer assisted multivariate analysis of the resulting high volume data (8000 data points) was performed. This computational approach enabled presentation of data in a variety of easily interpretable formats ( e.g., correlation tables and heat maps). Importantly, this computer aided approach was instrumental for the evaluation of assay accuracy, sensitivity, specificity, and robustness during the study. Crucial pieces of information were obtained to make timely adjustments for assay refinement. This progressive approach to developing an implementation-ready clinical assay, facilitated by computational analysis, produced a highly efficient, accurate and dependable serodiagnostics system. This system has effectively replaced the current state-of-the-art methodology (ELISA) used in mouse colony health management at the University of California and the Jackson Laboratory. A pathway to develop multiplex serology tests for infectious disease diagnosis described here serves as a model for multiplex immunoassay design, clinical validation, refinement and implementation.