Use Of Multiplex Assays Research Articles

Estimating the prevalence of two or more diseases using outcomes from multiplex group testing.

When screening a population for infectious diseases, pooling individual specimens (e.g., blood, swabs, urine, etc.) can provide enormous cost savings when compared to testing specimens individually. In the biostatistics literature, testing pools of specimens is commonly known as group testing or pooled testing. Although estimating a population-level prevalence with group testing data has received a large amount of attention, most of this work has focused on applications involving a single disease, such as human immunodeficiency virus. Modern methods of screening now involve testing pools and individuals for multiple diseases simultaneously through the use of multiplex assays. Hou etal. (2017, Biometrics, 73, 656-665) and Hou etal. (2020, Biostatistics, 21, 417-431) recently proposed group testing protocols for multiplex assays and derived relevant case identification characteristics, including the expected number of tests and those which quantify classification accuracy. In this article, we describe Bayesian methods to estimate population-level disease probabilities from implementing these protocols or any other multiplex group testing protocol which might be carried out in practice. Our estimation methods can be used with multiplex assays for two or more diseases while incorporating the possibility of test misclassification for each disease. We use chlamydia and gonorrhea testing data collected at the State Hygienic Laboratory at the University of Iowa to illustrate our work. We also provide an online R resource practitioners can use to implement the methods in thisarticle.

Epitope Tag Conjugation System Provides Versatility for Multiplex Assay Design

Abstract With the ever-growing use of multiplex assays in research, therapeutics, and diagnostics, the need to conjugate antibodies to dyes, enzymes, and other materials is increasing. While there are many challenges to overcome regarding conjugation, the largest hurdle is maintaining antibody activity after conjugation. To combat these conjugation challenges, Alpha Thera developed a conjugation system that utilizes a small adapter protein to label antibodies quickly and site-specifically, resulting in antibodies that maintain their activity post conjugation. The adapter protein contains a customizable C-terminal region that can be modified to contain diverse labels including fluorescent dyes, click-chemistry, drugs, and epitope tags. Incorporating the epitope tag conjugation system with Fortis Life Sciences anti-epitope tag detection antibodies, we developed an easy and adaptable system for generating conjugated antibodies to use in multiplex assays. We used this system to detect staining of multiple makers including CD3, TIM3, PCNA, and actin via flow cytometry, IHC, ICC, and western blot. One of the tested antibodies retained normal activity post-conjugation even though prior attempts with metal tagging via sulfhydryl groups resulted in lost activity. The vast array of conjugated anti-epitope tag antibodies allows for the selection of specific dyes/materials that are optimized to your desired assays. Additionally, since the secondary antibody is detecting the epitope tag, multiplexing is simplified, and antibody host is rendered irrelevant. This epitope tag system provides a simple, site-specific conjugation method that has incredible adaptability for any multiplex assay.

Informative array testing with multiplex assays

High-volume testing of clinical specimens for sexually transmitted diseases is performed frequently by a process known as group testing. This algorithmic process involves testing portions of specimens from separate individuals together as one unit (or "group") to detect diseases. Retesting is performed on groups that test positively in order to differentiate between positive and negative individual specimens. The overall goal is to use the least number of tests possible across all individuals without sacrificing diagnostic accuracy. One of the most efficient group testing algorithms is array testing. In its simplest form, specimens are arranged into a grid-like structure so that row and column groups can be formed. Positive-testing rows/columns indicate which specimens to retest. With the growing use of multiplex assays, the increasing number of diseases tested by these assays, and the availability of subject-specific risk information, opportunities exist to make this testing process even more efficient. We propose specific specimen arrangements within an array that can reduce the number of retests needed when compared with other array testing algorithms. We examine how to calculate operating characteristics, including the expected number of tests and the SD for the number of tests, and then subsequently find a best arrangement. Our methods are illustrated for chlamydia and gonorrhea detection with the Aptima Combo 2 Assay. We also provide R functions to make our research accessible to laboratories.

Soluble cytokine receptor levels in aqueous humour of patients with specific autoimmune uveitic entities: sCD30 is a biomarker of granulomatous uveitis.

PurposeSoluble cytokine receptors are potential biomarkers for immune activation and have a promising potential as immunotherapeutic agents. We investigated the levels of soluble cytokine receptors in aqueous humour (AH) samples from patients with specific autoimmune uveitic entities.MethodsPatients with active uveitis associated with Behçet’s disease (BD) (n = 13), sarcoidosis (n = 8), HLA-B27-related inflammation (n = 12), Vogt–Koyanagi–Harada (VKH) disease (n = 12) and control subjects (n = 9) were included. AH samples were analyzed with the use of multiplex assays for the proinflammatory cytokine tumour necrosis factor (TNF)-α and the soluble cytokine receptors sCD30, sCD163, sgp130, sIL-6 receptor-α (sIL-6R), sTNFRI and sTNFRII.ResultsTNF-α and soluble cytokine receptor AH levels were significantly higher in uveitis patients (n = 45) compared with controls (n = 9). When nongranulomatous uveitis (BD and HLA-B27-associated uveitis) was compared with granulomatous uveitis (sarcoidosis and VKH disease), the levels of sCD30 and sTNFRI/TNF-α and sTNFRII/TNF-α ratios were significantly enhanced in granulomatous uveitis. Finally, when comparing the profile in the specific uveitis entities, sCD30 levels were highest in patients with VKH disease. sgp130, sCD163, sIL-6R, sTNFRI and sTNFRII did not differ significantly between the four different clinical uveitic subgroups.ConclusionsSoluble cytokine receptors are significantly upregulated in autoimmune uveitis. CD30+ T cells might contribute to the inflammatory process in granulomatous uveitis, particularly in VKH disease. Granulomatous uveitis is also characterized by significantly higher sTNFRs/TNF-α ratios than nongranulomatous uveitis.

Regression analysis and variable selection for two-stage multiple-infection group testing data.

Group testing, as a cost-effective strategy, has been widely used to perform large-scale screening for rare infections. Recently, the use of multiplex assays has transformed the goal of group testing from detecting a single disease to diagnosing multiple infections simultaneously. Existing research on multiple-infection group testing data either exclude individual covariate information or ignore possible retests on suspicious individuals. To incorporate both, we propose a new regression model. This new model allows us to perform a regression analysis for each infection using multiple-infection group testing data. Furthermore, we introduce an efficient variable selection method to reveal truly relevant risk factors for each disease. Our methodology also allows for the estimation of the assay sensitivity and specificity when they are unknown. We examine the finite sample performance of our method through extensive simulation studies and apply it to a chlamydia and gonorrhea screening data set to illustrate its practical usefulness.

Array testing for multiplex assays.

Group testing involves pooling individual specimens (e.g., blood, urine, swabs, etc.) and testing the pools for the presence of disease. When the proportion of diseased individuals is small, group testing can greatly reduce the number of tests needed to screen a population. Statistical research in group testing has traditionally focused on applications for a single disease. However, blood service organizations and large-scale disease surveillance programs are increasingly moving towards the use of multiplex assays, which measure multiple disease biomarkers at once. Tebbs and others (2013, Two-stage hierarchical group testing for multiple infections with application to the Infertility Prevention Project. Biometrics69, 1064-1073) and Hou and others (2017, Hierarchical group testing for multiple infections. Biometrics73, 656-665) were the first to examine hierarchical group testing case identification procedures for multiple diseases. In this article, we propose new non-hierarchical procedures which utilize two-dimensional arrays. We derive closed-form expressions for the expected number of tests per individual and classification accuracy probabilities and show that array testing can be more efficient than hierarchical procedures when screening individuals for multiple diseases at once. We illustrate the potential of using array testing in the detection of chlamydia and gonorrhea for a statewide screening program in Iowa. Finally, we describe an R/Shiny application that will help practitioners identify the best multiple-disease case identification algorithm.

The role of the NxTAG® respiratory pathogen panel assay and other multiplex platforms in clinical practice

ABSTRACTIntroduction: The advent of nucleic acid amplification tests has significantly improved the aetiologic diagnosis of respiratory infections. However, multiplex real-time polymerase chain reaction (PCR) can be technologically challenging.Areas covered: This paper reports the results of the main published studies on the NxTAG Respiratory Pathogen Panel (RPP) and discusses the advantages and disadvantages of extensive use of multiplex assays in clinical practice.Expert commentary: Currently available data seem to indicate that routine use of multiplex assays, including NxTAG RPP Assay, should be recommended only when epidemiological data concerning circulation of viruses and bacteria have to be collected. Their use in clinical practice seems debatable. They have limited sensitivity and specificity at least in the identification of some infectious agents or, as in the case of NxTAG RPP, they have not been evaluated in a sufficient number of patients to allow definitive conclusions. In the future, the clinical relevance of multiplex assays, including NxTAG RPP, could significantly increase, mainly because a number of new antiviral agents effective against several respiratory viruses for which no drug is presently available will be marketed. In addition, it is highly likely that the efficiency of multiplex assays will be significantly improved.

Biomarkers of spontaneous preterm birth: a systematic review of studies using multiplex analysis.

Despite decades of research on risk indicators of spontaneous preterm birth (PTB), reliable biomarkers are still not available to screen or diagnose high-risk pregnancies. Several biomarkers in maternal and fetal compartments have been mechanistically linked to PTB, but none of them are reliable predictors of pregnancy outcome. This systematic review was conducted to synthesize the knowledge on PTB biomarkers identified using multiplex analysis. Three electronic databases (PubMed, EMBASE and Web of Science) were searched for studies in any language reporting the use of multiplex assays for maternal biomarkers associated with PTB published from January 2005 to March 2014. Retrieved citations (3631) were screened, and relevant studies (33) were selected for full-text reading. Ten studies were included in the review. Forty-two PTB-related proteins were reported, and RANTES and IL-10 (three studies) followed by MIP-1β, GM-CSF, Eotaxin, and TNF-RI (two studies) were reported more than once in maternal serum. However, results could not be combined due to heterogeneity in type of sample, study population, assay, and analysis methods. By this systematic review, we conclude that multiplex assays are a potential technological advancement for identifying biomarkers of PTB, although no single or combination of biomarkers could be identified to predict PTB risk.

Inflammatory cytokine response following acute tibial plateau fracture.

The objective of the present study was to evaluate human synovial fluid for inflammatory cytokine concentrations following acute tibial plateau fracture. Our hypothesis was that there would be an elevated inflammatory response following intra-articular fracture, and that the inflammatory response would be greater after high-energy compared with low-energy injuries. Between December 2011 and June 2013, we prospectively enrolled forty-five patients with an acute tibial plateau fracture. Synovial fluid aspirations were performed on the injured and uninjured knees. Twenty patients who required an external fixator followed by delayed fixation underwent aspiration at both surgical procedures. The concentrations of interferon-gamma (IFN-γ), interleukin-1 beta (IL-1β), interleukin-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12(p70), IL-13, IL-17A, tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1β) were quantified with use of multiplex assays. The forty-five patients had an average age of forty-two years (range, twenty to sixty years). There were twenty-four low-energy and twenty-one high-energy tibial plateau injuries. There was a significant difference between injured and uninjured knees (p < 0.001) with regard to concentrations of IL-1β, IL-6, IL-8, IL-10, IL-1RA, and MCP-1. There was not a detectable difference in synovial fluid cytokine concentrations between high and low-energy injuries. The concentrations of IL-10 (p < 0.001), IL-1RA (p = 0.002), IL-6 (p < 0.001), IL-8 (p < 0.001), and MCP-1 (p = 0.002) were significantly greater in the injured knee than in the uninjured knee at the second aspiration, at a mean of 9.5 days (range, three to twenty-one days) after the initial injury. There was a significant local inflammatory response following acute tibial plateau fracture. There was not a detectable difference in inflammatory cytokine concentration between high and low-energy injuries. Synovial fluid concentrations of IL-10, IL-8, IL-6, IL-1RA, and MCP-1 remained elevated at the second aspiration.

Multiplex PCR to Detect T. gondii Infection based on B1 Gene and 529 bp Repetitive Element

Polymerase Chain Reaction (PCR) has made a significant improvement in the diagnosis of toxoplasmosis. Nevertheless, a wide variety of targets and primers has been evaluated in different independent assays, and only few comparative studies have been performed. Even when the B1 gene and the 529 repetitive element have been the most studied markers, there no concluding remarks respect the best. Objetive: The aim of the present study was to design a multiplex PCR assay to simultaneously detect these two markers in a single multiplex PCR to diagnose T. gondii infection. Methods: Specific PCR primers targeting the B1 gene and 529 repetitive element were evaluated. After a careful optimization of multiplex PCR process, analytical sensitivity and specificity were evaluated. The usefulness of multiplex assay was evaluated using DNA from 8 T. gondii reference strains, corresponding to the three principal genotypes, and 70 clinical samples (blood) from AIDS patients with and without Toxoplasmic encephalitis (TE). Results: Our preliminar results show that multiplex PCR assay is able to amplify both targets in using DNA from one parasite in the mix PCR reaction. Unspecific reactions for other microorganisms were not observed. The diagnostic sensitivity of multiplex assay in blood, according to the Centers for Disease Control and Prevention (CDC) criteria, was 86.6% while the diagnostic specificity was 100%. Only one sample was positive to one marker (B1 gene) in the multiplex reaction. Conclusion: This multiplex PCR method is the first multiplex PCR evaluating the detection of T. gondii DNA in TE cases and it proved to be rapid, enough sensitive, highly specific and inexpensive respect to perform independent assays for each marker, real time PCR or nested PCR assays.

Luminex and other multiplex high throughput technologies for the identification of, and host response to, environmental triggers of type 1 diabetes.

Complex interactions between a series of environmental factors and genes result in progression to clinical type 1 diabetes in genetically susceptible individuals. Despite several decades of research in the area, these interactions remain poorly understood. Several studies have yielded associations of certain foods, infections, and immunizations with the onset and progression of diabetes autoimmunity, but most findings are still inconclusive. Environmental triggers are difficult to identify mainly due to (i) large number and complex nature of environmental exposures, including bacteria, viruses, dietary factors, and environmental pollutants, (ii) reliance on low throughput technology, (iii) less efforts in quantifying host response, (iv) long silent period between the exposure and clinical onset of T1D which may lead to loss of the exposure fingerprints, and (v) limited sample sets. Recent development in multiplex technologies has enabled systematic evaluation of different classes of molecules or macroparticles in a high throughput manner. However, the use of multiplex assays in type 1 diabetes research is limited to cytokine assays. In this review, we will discuss the potential use of multiplex high throughput technologies in identification of environmental triggers and host response in type 1 diabetes.

Comparative analysis of IgG responses to Plasmodium falciparum MSP1p19 and PF13-DBL1α1 using ELISA and a magnetic bead-based duplex assay (MAGPIX®-Luminex) in a Senegalese meso-endemic community.

BackgroundNumerous Plasmodium falciparum antigens elicit humoral responses in humans living in endemic areas. Use of multiplex assays is a convenient approach to monitor the antibody response against multiple antigens, but to integrate multiplex assay-derived data with datasets, generated previously using ELISA, comparative studies are needed. This work compares antibody responses to two P. falciparum antigens monitored using both technologies.MethodsThe IgG response against the merozoite surface protein-1 PfMSP1p19 and the PF13-DBL1α1 domain of the P. falciparum Erythrocyte Membrane Protein1, expressed by the rosette-forming parasite 3D7/PF13 (PF13), was investigated using ELISA and a MAGPIX®-Luminex duplex assay. Archived plasma samples collected before the rainy season from 217 villagers living in Ndiop, a Senegalese meso-endemic setting, were studied. ROC analysis was used to define the optimal antibody measure readout. Association of antibody levels with protection against clinical malaria was analysed using Poisson regression in a retrospective study from active case detection records performed during the 5.5-month transmission season that followed blood sampling.ResultsThere was a strong positive correlation (P <10-3) between ELISA and MAGPIX®-Luminex-MFI (median fluorescence intensity) values for antibody to PfMSP1p19 (rho = 0.78) and PF13-DBL1α1 (rho = 0.89), with a similar degree of concordance in all age groups. Antibody levels to both antigens were high but displayed a different age-associated pattern. Independent age-adjusted Poisson regression analysis showed a significant association with protection only for IgG responses to MSP1p19 (P <0.01 RR = 0.71 [0.53-0.93]) measured by ELISA.ConclusionThe individual ELISA and duplex-MAGPIX assays provide a concordant evaluation of age-associated antibody responses to MSP1p19 and PF13-DBL1α1, irrespective of the formulation of antibody levels (values, ratios or ROC-adjusted figures) but do diverge with regard to the association of antibody levels with clinical protection in age-adjusted models. This may reflect incomplete overlap of the epitopes presented in the two formats. Further development for multiplex assessment of antibody responses to a larger panel of antigens with the robust and cost effective MAGPIX®-Luminex technology is warranted.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-410) contains supplementary material, which is available to authorized users.

Analysis of tear fluid proteins: use of multiplex assays in profiling biomarkers of dry eye disease

Analysis of tear fluid proteins: use of multiplex assays in profiling biomarkers of dry eye disease

Analysis of tear fluid proteins: use of multiplex assays in profiling biomarkers of dry eye disease

Aims Dry eye disease (DED) is a distressing condition that is commonly associated with ageing, contact lens wear and autoimmune disease. DED is one of the fastest-growing eye disorders in older populations (over-50s) and is significantly underdiagnosed. Multiplex studies of tear fluids from DED patient have suggested a role for inflammatory cytokines in its pathology. In this study, we investigated tear fluids from DED and normal subjects for a panel of 7 cytokines, using the multiplex bead assay.