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Pharmaceutical and biotechnology companies are increasingly being challenged to shorten the cycle time between design, make, test, and analyze (DMTA) compounds. Automation of multiplex assays to obtain multiparameter data on the same robotic run is instrumental in reducing cycle time. Consequently, an increasing need in automated systems to streamline laboratory workflows with the goal to expedite assay cycle time and enhance productivity has grown in industrial and academic institutions in the past decades. Herein, we present a customized robotic platform with operational modularity and flexibility, designed to automate entire assay workflows involving multistep reagent dispensing, mixing, lidding/de-lidding, incubation, centrifugation, and final readout steps by linking spinnaker robot with various peripheral instruments. Compared to manual workflows, the system can seamlessly execute processes with high efficiency, evaluated by standard assay validation protocols for robustness and reproducibility. Furthermore, the system can perform multiple, independent protocols in parallel, and has high-throughput capacity. In this publication, we demonstrate that the modular robotic platform can fully automate multiplex assay workflows through ‘one-click-and-run’ solution with tremendous benefits in liberating manual intervention, boosting productivity while producing high-quality data combined with reduced cycle time (>20 %), as well as expanding the capacity for higher throughput.

Abstract Background Cytokines/chemokines play essential roles in the occurrence and progression of hepatitis B virus (HBV) infection. This study aimed to observe the expression patterns of 10 related cytokines/chemokines in the serum of healthy individuals, self-limited patients and HBV-infected patients at different stages of disease (chronic hepatitis B (CHB), liver cirrhosis (LC), hepatocellular dysplastic nodules (DNs) and hepatocellular carcinoma (HCC)) and to analyze the relationships of these cytokines/chemokines with disease progression. Methods The levels of six cytokines (FGF-2, IFN-α2, IL-4, IL-6, IL-10 and VEGF-A) and four chemokines (GRO-α, IL-8, IP-10 and MCP-1) were quantified using Luminex multiplex technology. Results There were no significant differences in the expression of the 10 cytokines/chemokines between healthy individuals and self-limited patients. The levels of IL-4, IL-6, and IL-8 increased significantly in the CHB and LC groups. IL-10 was highly expressed in the HCC group. The level of IP-10 was significantly greater in all liver disease groups (CHB, LC, DN and HCC) than in the HI and SL-HBV groups, while the level of GRO was significantly lower in all liver disease groups than in the HI and SL-HBV groups. The levels of the 10 cytokines/chemokines were not significantly different between the preoperative group and the two-day postoperative group. Significant increases in the levels of IL-4, VEGF-A and IL-8 and significant decreases in those of IL-10 and GRO-α were observed 3 months after surgery. Correlation analysis revealed that most of the cytokines/chemokines with significant correlation differences were positively correlated before and after HCC surgery. Conclusion Our results highlight the fluctuating status of specific cytokines in HBV infection-related disease progression. It is speculated that these cytokines may be used as serum markers to monitor dynamic changes during the progression of HBV-related liver disease and to predict patient prognosis.

Background: Cytokines/chemokines play essential roles in the occurrence and progression of hepatitis B virus (HBV) infection. This study aimed to observe the expression patterns of 10 related cytokines/chemokines in the serum of healthy individuals, self-limited patients and HBV-infected patients at different stages of disease (chronic hepatitis B (CHB), liver cirrhosis (LC), hepatocellular dysplastic nodules (DNs) and hepatocellular carcinoma (HCC)) and to analyze the relationships of these cytokines/chemokines with disease progression. Methods: The levels of six cytokines (FGF-2, IFN-α2, IL-4, IL-6, IL-10 and VEGF-A) and four chemokines (GRO-α, IL-8, IP-10 and MCP-1) were quantified using Luminex multiplex technology. Results: There were no significant differences in the expression of the 10 cytokines/chemokines between healthy individuals and self-limited patients. The levels of IL-4, IL-6, and IL-8 increased significantly in the CHB and LC groups. IL-10 was highly expressed in the HCC group. The level of IP-10 was significantly greater in all liver disease groups (CHB, LC, DN and HCC) than in the HI and SL-HBV groups, while the level of GRO was significantly lower in all liver disease groups than in the HI and SL-HBV groups. The levels of the 10 cytokines/chemokines were not significantly different between the preoperative group and the two-day postoperative group. Significant increases in the levels of IL-4, VEGF-A and IL-8 and significant decreases in those of IL-10 and GRO-α were observed 3 months after surgery. Correlation analysis revealed that most of the cytokines/chemokines with significant correlation differences were positively correlated before and after HCC surgery. Conclusion: Our results highlight the fluctuating status of specific cytokines in HBV infection-related disease progression. It is speculated that these cytokines may be used as serum markers to monitor dynamic changes during the progression of HBV-related liver disease and to predict patient prognosis. [ABSTRACT FROM AUTHOR]

Antibody response on polysaccharide- and protein-based vaccines is useful to test B cell functionality. As only few studies have explored the value of studying immune response to both vaccines, we evaluated the clinical value of anti-polysaccharide and anti-protein Luminex-based multiplex assays in context of primary immunodeficiency (PID) diagnosis. A 10-plex Luminex-based assay detecting antibodies to ten pneumococcal polysaccharide (PnPS) serotypes [present in unconjugated Pneumovax, not in 13-valent pneumococcal conjugated vaccine (PCV)] and a 5-plex assay detecting antibodies to five protein antigens (present in DTap/Tdap) were clinically validated in healthy individuals (n=99) and in retrospective (n=399) and prospective (n=108) patient cohorts. Clinical features of individuals with impaired response to PnPS and/or proteins were compared to those with normal response. Antigen-specific antibody thresholds were determined in healthy individuals. Individuals with impaired anti-PnPS responses and deficient immunoglobulin levels suffered more from autoimmune diseases and had lower B cell levels compared to individuals with impaired anti-PnPS response with normal immunoglobulin levels. Individuals with combined impaired response to PnPS and proteins showed more severe clinical manifestations compared to individuals with isolated impaired response to PnPS or proteins. Eight of the 11 individuals with severely impaired responses to both PnPS and proteins had common variable immunodeficiency. Evaluation of the anti-PnPS response to four serotypes not contained in 20-valent PCV was comparable to evaluation to ten serotypes not contained in 13-valent PCV. Multiplexed assessment of anti-PnPS and anti-protein responses combined with immunoglobulin quantification provides useful clinical information to support PID diagnosis. [ABSTRACT FROM AUTHOR]

Malaria remains a major global health challenge, causing over 0.6 million yearly deaths. To understand naturally acquired immunity in adult human populations in malaria-prevalent regions, improved serological tools are needed, particularly where multiple malaria parasite species co-exist. Slide-based and bead-based multiplex approaches can help characterize antibodies in malaria patients from endemic regions, but these require pure, well-defined antigens. To efficiently bypass purification steps, codon-optimized malaria antigen genes with N-terminal FLAG-tag and C-terminal Ctag sequences were expressed in a wheat germ cell-free system and adsorbed on functionalized BioPlex beads. In a pilot study, 15 P. falciparum antigens, 8 P. vivax antigens, and a negative control (GFP) were adsorbed individually on functionalized bead types through their Ctag. To validate the multiplexing powers of this platform, 10 P. falciparum-infected patient sera from a US NIH MESA-ICEMR study site in Goa, India, were tested against all 23 parasite antigens. Serial dilution of patient sera revealed variations in potency and breadth of antibodies to various parasite antigens. Individual patients revealed informative variations in immunity to P. falciparum versus P. vivax. This multiplex approach to malaria serology captures varying immunity to different human malaria parasite species and different parasite antigens. This approach can be scaled to track the dynamics of antibody production during one or more human malaria infections. [ABSTRACT FROM AUTHOR]

Biosensors are nowadays being used in various fields including disease diagnosis and clinical analysis. The ability to detect biomolecules associated with disease is vital not only for accurate diagnosis of disease but also for drug discovery and development. Among the different types of biosensors, electrochemical biosensor is most widely used in clinical and health care services especially in multiplex assays due to its high susceptibility, low cost and small in size. This article includes comprehensive review of biosensors in medical field with special emphasis on electrochemical biosensors for multiplex assays and in healthcare services. Also, the publications on electrochemical biosensors are increasing rapidly; therefore, it is crucial to be aware of any latest developments or trends in this field of research. We used bibliometric analyses to summarize the progress of this research area. The study includes global publication counts on electrochemical biosensors for healthcare along with various bibliometric data analyses by VOSviewer software. The study also recognizes the top authors and journals in the related area, and determines proposal for monitoring research. [ABSTRACT FROM AUTHOR]

Biosensors are nowadays being used in various fields including disease diagnosis and clinical analysis. The ability to detect biomolecules associated with disease is vital not only for accurate diagnosis of disease but also for drug discovery and development. Among the different types of biosensors, electrochemical biosensor is most widely used in clinical and health care services especially in multiplex assays due to its high susceptibility, low cost and small in size. This article includes comprehensive review of biosensors in medical field with special emphasis on electrochemical biosensors for multiplex assays and in healthcare services. Also, the publications on electrochemical biosensors are increasing rapidly; therefore, it is crucial to be aware of any latest developments or trends in this field of research. We used bibliometric analyses to summarize the progress of this research area. The study includes global publication counts on electrochemical biosensors for healthcare along with various bibliometric data analyses by VOSviewer software. The study also recognizes the top authors and journals in the related area, and determines proposal for monitoring research.

Malaria remains a major global health challenge, causing over 0.6 million yearly deaths. To understand naturally acquired immunity in adult human populations in malaria-prevalent regions, improved serological tools are needed, particularly where multiple malaria parasite species co-exist. Slide-based and bead-based multiplex approaches can help characterize antibodies in malaria patients from endemic regions, but these require pure, well-defined antigens. To efficiently bypass purification steps, codon-optimized malaria antigen genes with N-terminal FLAG-tag and C-terminal Ctag sequences were expressed in a wheat germ cell-free system and adsorbed on functionalized BioPlex beads. In a pilot study, 15 P. falciparum antigens, 8 P. vivax antigens, and a negative control (GFP) were adsorbed individually on functionalized bead types through their Ctag. To validate the multiplexing powers of this platform, 10 P. falciparum-infected patient sera from a US NIH MESA-ICEMR study site in Goa, India, were tested against all 23 parasite antigens. Serial dilution of patient sera revealed variations in potency and breadth of antibodies to various parasite antigens. Individual patients revealed informative variations in immunity to P. falciparum versus P. vivax. This multiplex approach to malaria serology captures varying immunity to different human malaria parasite species and different parasite antigens. This approach can be scaled to track the dynamics of antibody production during one or more human malaria infections.

Pharmaceutical and biotechnology companies are increasingly being challenged to shorten the cycle time between design, make, test, and analyze (DMTA) compounds. Automation of multiplex assays to obtain multiparameter data on the same robotic run is instrumental in reducing cycle time. Consequently, an increasing need in automated systems to streamline laboratory workflows with the goal to expedite assay cycle time and enhance productivity has grown in industrial and academic institutions in the past decades. Herein, we present a customized robotic platform with operational modularity and flexibility, designed to automate entire assay workflows involving multistep reagent dispensing, mixing, lidding/de-lidding, incubation, centrifugation, and final readout steps by linking spinnaker robot with various peripheral instruments. Compared to manual workflows, the system can seamlessly execute processes with high efficiency, evaluated by standard assay validation protocols for robustness and reproducibility. Furthermore, the system can perform multiple, independent protocols in parallel, and has high-throughput capacity. In this publication, we demonstrate that the modular robotic platform can fully automate multiplex assay workflows through 'one-click-and-run' solution with tremendous benefits in liberating manual intervention, boosting productivity while producing high-quality data combined with reduced cycle time (>20 %), as well as expanding the capacity for higher throughput., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Immunoassays are a powerful diagnostic tool and are widely used for the quantification of proteins and biomolecules in medical diagnosis and research. Enzyme-linked immunosorbent assay (ELISA) is the most commonly used immunoassay format and allows the detection of biomarkers at a very low concentration. The diagnostic platforms such as enzyme immunoassay (EIA), chemiluminescence (CL) assay, polymerase chain reaction (PCR), flow cytometry (FC), and mass spectrometry (MS) have been used to identify molecular biomarkers. However, these diagnostic tools requiring expensive equipment, long testing time, and qualified personnel that is not always available in small local hospitals with limited resources. The lateral flow immunoassay (LFIA) platform was developed for rapidly obtaining laboratory results and to make urgent decisions in emergency medicine, as well as the recently introduced concept of testing at the site of care (point-of-care, POC). The simultaneous measurement of different substances from a single sample called multiplex assays have become increasingly significant for in vitro quantification of multiple analytes in a single sample, thereby minimising cost, time, and volume. In multiplex immunoassays, the ligands are immobilized either in planar format (flat surface) or on microspheres in suspension that binds to target analytes in sample. The multiplex technology has established itself in proteomic networks and pathways, validation of genomic discoveries, and in the development of clinical biomarkers. In the present review article, various types of monoplex/simplex and complex/multiplex immunoassays have been analysed that are increasingly being applied in laboratory medicine. Also, some advantages and disadvantages of these multiplex assays have also been included such as experimental animals, in vitro tests using cell lines and tissue samples, 3-dimensional modelling and bioprinting, in silico tests, organ-on-chip, and computer modelling. [ABSTRACT FROM AUTHOR]

Introduction: With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold-standard reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. This study shows steps on how to develop different ddPCR SAR-CoV-2 assays including higher order multiplex assays for SARS-CoV-2 detection and antiviral screening. Methods: Using multiple primer/probe sets, we developed, optimized, and analyzed the performance of simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and quadruplex (4 targets) SARS-CoV-2 ddPCR assays based on a two-color ddPCR detection system. Results: Results showed that the quadruplex assay had similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical samples demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug could not. Conclusion: Our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude-based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications. [ABSTRACT FROM AUTHOR]

There exists a close relationship between cardiovascular diseases and chronic kidney disease. Apolipoprotein A1 and high-density lipoprotein (HDL) cholesterol are widely used as cardiovascular risk markers but they also have anti-inflammatory properties. The aim of this study was to investigate any associations between HDL levels and cytokine levels in urine. We randomly selected 90 urine samples from the Prospective Investigation of the Vasculature in Uppsala Seniors Study (41 males and 49 females). The samples were analyzed with 2 multiplex assays, Multiplex Inflammation I and Cardiovascular II kits (Olink Bioscience, Uppsala, Sweden). We analyzed the correlations between 158 cytokines in urine with apolipoprotein A1, HDL cholesterol, apolipoprotein B, and low-density lipoprotein cholesterol. There were strong correlations for apolipoprotein A1 and HDL cholesterol with individual cytokines. After adjustment for multiplicity testing, there were 33 significant correlations between apolipoprotein A1 and cytokine levels and 14 of these were also significantly correlated with HDL cholesterol. The strongest associations were observed for IL-1α, SPON2, RAGE, PAR-1, TRAIL-R2, IL-4RA, TNFRSF11A, and SCF. A total of 28 out of 33 correlations were negative, indicating a negative relationship between apolipoprotein A1 and urinary cytokines. The study shows a negative correlation between apolipoprotein A1 and HDL cholesterol and urinary cytokine levels. The finding is in agreement with the anti-inflammatory properties of HDL. [ABSTRACT FROM AUTHOR]

Abstract Background Plasmodium falciparum infections are especially severe in pregnant women because infected erythrocytes (IE) express VAR2CSA, a ligand that binds to placental trophoblasts, causing IE to accumulate in the placenta. Resulting inflammation and pathology increases a woman’s risk of anemia, miscarriage, premature deliveries, and having low birthweight (LBW) babies. Antibodies (Ab) to VAR2CSA reduce placental parasitaemia and improve pregnancy outcomes. Currently, no single assay is able to predict if a woman has adequate immunity to prevent placental malaria (PM). This study measured Ab levels to 28 malarial antigens and used the data to develop statistical models for predicting if a woman has sufficient immunity to prevent PM. Methods Archival plasma samples from 1377 women were screened in a bead-based multiplex assay for Ab to 17 VAR2CSA-associated antigens (full length VAR2CSA (FV2), DBL 1-6 of the FCR3, 3D7 and 7G8 lines, ID1-ID2a (FCR3 and 3D7) and 11 antigens that have been reported to be associated with immunity to P. falciparum (AMA-1, CSP, EBA-175, LSA1, MSP1, MSP2, MSP3, MSP11, Pf41, Pf70 and RESA)). Ab levels along with clinical variables (age, gravidity) were used in the following seven statistical approaches: logistic regression full model, logistic regression reduced model, recursive partitioning, random forests, linear discriminant analysis, quadratic discriminant analysis, and support vector machine. Results The best and simplest model proved to be the logistic regression reduced model. AMA-1, MSP2, EBA-175, Pf41, and MSP11 were found to be the top five most important predictors for the PM status based on overall prediction performance. Conclusions Not surprising, significant differences were observed between PM positive (PM+) and PM negative (PM−) groups for Ab levels to the majority of malaria antigens. Individually though, these malarial antigens did not achieve reasonably high performances in terms of predicting the PM status. Utilizing multiple antigens in predictive models considerably improved discrimination power compared to individual assays. Among seven different classifiers considered, the reduced logistic regression model produces the best overall predictive performance.

Background: Based on advances over several decades, a significant number of autoantibodies are aiding the diagnosis, differential diagnosis and even prognostic estimates of patients with connective tissue diseases (CTD). Based on cost and time constraints, multiplex assays are used more and more in routine practice. Here we describe the evaluation results of a novel spot immunoassay (SeraSpot® ANA) for multiplex analysis of the main CTD specific autoantibodies, which is based on autoantigens immobilized on microtitration plates. Methods: For evaluation of the ANA spot immunoassay, comprising dsDNA, histone, nucleosome, Scl-70, U1-RNP (mixture of U1-RNP-A, C, and 70k), Sm (SmD), RibP (P0), Ro52/TRIM21, Ro60, La/SS-B, CENP-B, Jo-1, PM/Scl-100, and Ku as target antigens, sera of 489 patients with systemic autoimmune rheumatic disease (SARD), sera of 54 patients with herpes virus infections, and sera of 202 apparently healthy individuals (AHI) were tested. Results: The concordance between the SeraSpot and EIA results of disease specific autoantibodies (AABs) was high (94 - 97% for CENP-B, Jo-1, Scl-70, Sm, La, Ro52 and Ro60 antibodies) to moderate (86.7% for dsDNA antibodies), and no major differences in the frequency of these AABs in patients with CTD were observed. In SLE patients, the SeraSpot results of dsDNA antibodies correlate better with CLIFT results and HEp-2 cell pattern than the EIA results. The diagnostic specificities of SLE, SSc, SjS, and myositis specific AABs are very high (96.5 - 100%) compared to apparently healthy individuals, but lower with regard to serological differentiation of the SARD entities (86.6 - 99.1%). However, very high positive likelihood ratios (10 up to infinite) could be obtained by disease specific AAB profiles. Conclusions: The SeraSpot® ANA assay allows the detection of 14 AAB specificities used for the diagnosis and differentiation of CTD in one approach. Although the concordance between the SeraSpot® assay and EIA is moderate for most of the tested AAB specificities, defined AAB profiles are suitable for the correct diagnosis of the CTD entities. If time matters, the authors suggest the parallel employment of immunofluorescence on HEp-2 cells and the SeraSpot® ANA assay for screening and specific AAB determination of patients with suspected CTD. [ABSTRACT FROM AUTHOR]

Abstract Cell-based assays against cell surface receptor targets are essential in vitro models of target-based drug discovery. At the lead generation phase large-scale functional screening assays monitoring individual cellular readouts detect interactions between the compounds and the predefined pathways but might lack sufficient sensitivity owing to the complexity of downstream signaling pathways. Cellular label-free assays offer advantages over labeled detection approaches as they reflect whole-cell responses without the prerequisite of detecting only a single cellular analyte and introducing additional genetic manipulations in favor of the chosen detection method. The combination of a label-free assay and labeled assays might integrate the advantageous characteristics of both approaches with regards to added pharmacological information and a bigger pool of chemical starting material. Here we report multiplexing of dynamic mass redistribution label-free technology with HTRF-based cAMP detection on an alpha2c adrenergic receptor expressing cell line. Besides describing the challenging assay development work associated with the set goal, a pilot screening campaign on ca. 1600 compounds is also presented. The combined assay demonstrated the ability to detect relevant activities in both readouts. Interpretation of the results as well as an outlook for further possible opportunities and applications are also discussed. Highlights • Label-free DMR assay can be miniaturized. • HTRF cAMP assay is feasible on adherent cells in special label-free DMR plates. • Label-free DMR and the HTRF assay can be combined in a miniaturized duplex HTS assay. • This assay enables to detect the broadest repertoire of signaling events. • No need for genetic manipulation facilitating duplex HTS on more native cell systems. [ABSTRACT FROM AUTHOR]

Many ingredients in cosmetic products help to develop complex formulations that improve the quality of life in terms of disease prevention, health maintenance, beauty enhancement, and building self-esteem. The beneficial effects promoted from the use of biomolecule-rich substances into the formulations of various topical application products are considered useful ingredients in cosmetic and therapeutic applications. This review article attempts to understand the various biomolecules found in cosmetic products, particularly macromolecules, which may have an important role in prevention or preservation. Increasing demand of cosmetics all over the world has increased the awareness related to safety issue. Cosmetic products may contain potential contact allergens or precursors that can be oxidized or metabolized to generate contact allergens which can potentially cause allergic reactions or dermatitis. These substances can pose hazards to human health due to their ability to activate T cells that can cause allergic contact dermatitis, an inflammatory skin disease. Finally, the simultaneous on-site measurement of different substances from a single sample, called multiplexed point-of-care testing, has recently become increasingly important for the in vitro quantification of pathological or toxicological samples. Hence, the technological advancements in clinical sciences will be helpful in the identification of ingredients in cosmetic preparations. [ABSTRACT FROM AUTHOR]

To transform from reactive to proactive healthcare, there is an increasing need for low-cost and portable assays to continuously perform health measurements. The paper-based analytical devices could be a potential fit for this need. To miniaturize the multiplex paper-based microfluidic analytical devices and minimize reagent use, a fabrication method with high resolution along with low fabrication cost should be developed. Here, we present an approach that uses a desktop pen plotter and a high-resolution technical pen for plotting high-resolution patterns to fabricate miniaturized paper-based microfluidic devices with hundreds of detection zones to conduct different assays. In order to create a functional multiplex paper-based analytical device, the hydrophobic solution is patterned on the cellulose paper and the reagents are deposited in the patterned detection zones using the technical pens. We demonstrated the effect of paper substrate thickness on the resolution of patterns by investigating the resolution of patterns on a chromatography paper with altered effective thickness. As the characteristics of the cellulose paper substrate such as thickness, resolution, and homogeneity of pore structure affect the obtained patterning resolution, we used regenerated cellulose paper to fabricate detection zones with a diameter as small as 0.8 mm. Moreover, in order to fabricate a miniaturized multiplex paper-based device, we optimized packing of the detection zones. We also showed the capability of the presented method for fabrication of 3D paper-based microfluidic devices with hundreds of detection zones for conducting colorimetric assays.

Malaria remains a major global health challenge, causing over 0.6 million yearly deaths. To understand naturally acquired immunity in adult human populations in malaria-prevalent regions, improved serological tools are needed, particularly where multiple malaria parasite species co-exist. Slide-based and bead-based multiplex approaches can help characterize antibodies in malaria patients from endemic regions, but these require pure, well-defined antigens. To efficiently bypass purification steps, codon-optimized malaria antigen genes with N-terminal FLAG-tag and C-terminal Ctag sequences were expressed in a wheat germ cell-free system and adsorbed on functionalized BioPlex beads. In a pilot study, 15 P. falciparum antigens, 8 P. vivax antigens, and a negative control (GFP) were adsorbed individually on functionalized bead types through their Ctag. To validate the multiplexing powers of this platform, 10 P. falciparum -infected patient sera from a US NIH MESA-ICEMR study site in Goa, India, were tested against all 23 parasite antigens. Serial dilution of patient sera revealed variations in potency and breadth of antibodies to various parasite antigens. Individual patients revealed informative variations in immunity to P. falciparum versus P. vivax . This multiplex approach to malaria serology captures varying immunity to different human malaria parasite species and different parasite antigens. This approach can be scaled to track the dynamics of antibody production during one or more human malaria infections.

Evaluation of inter dopant electronic interactions is important to maximize photoluminescence for their use as multiplex assays. This work compares the photophysical properties of lanthanide dopants in cases where the lanthanide moieties have been either synthetically co-doped in nanoparticles or two singly doped nanoparticles were physically mixed post-synthetically. Nd 3+ − Sm 3+ and Nd 3+ − Er 3+ were considered as dopants in the TiO 2 nanoparticles. Remarkable differences in lanthanide emission have been observed between the two strategies of forming the multiple and distinct lanthanide containing assemblies. The inter lanthanide electronic interactions were found to be evident in the synthetic co-doping, with the same being absent in the physically mixed assembly. As a consequence, the Nd 3+ emission in near infrared (NIR) and Sm 3+ , Er 3+ emission in visible spectral range respectively have been found to be more pronounced in the physically mixed assembly as compared to that in the synthetically co-doped nanoparticles. The situation is however reversed for the Er 3+ emission in NIR spectral range. These findings collectively provide a facile way to maximize dopant emission and indicate the importance of tuning the inter lanthanide electronic interactions for better suitability as multiplex agent, in an as-desired way. [ABSTRACT FROM AUTHOR]

The collector sea urchin Tripneustes gratilla has been identified as a species with potential for aquaculture production in South Africa. The species’ roe is considered a culinary delicacy in Asia and Europe. However, T. gratilla remains genetically uncharacterised in South Africa. Therefore, the purpose of this study was to provide baseline genetic information consisting of estimates of genetic diversity and population stratification, which may aid in future sustainable use of this urchin resource. A total of 22 species-specific microsatellite markers were used for the genetic characterisation of T. gratilla samples from along the South African coast, at Haga Haga, Coffee Bay, Hibberdene, Ballito Bay and Sodwana Bay. A moderate level of genetic diversity was observed, with an average number of alleles of 7.89 and an average effective number of alleles of 6.57, as well as an average observed heterozygosity of 0.55. Population differentiation tests suggest that the geographically representative samples form part of a single, large interbreeding population, with a global Fst estimate of 0.02 (p > 0.05). This finding is likely explained by high levels of gene flow between these locations caused by extensive larval dispersal during the planktonic larval stage. The panmixia observed within these natural populations of T. gratilla indicate that they could be managed as a single genetic stock. [ABSTRACT FROM AUTHOR]

Cytokines are known to play important roles in a wide range of pathologic conditions spanning all organ systems in every species studied. As our knowledge of the physiology of individual cytokines expands and our ability to measure multiple cytokines in smaller biological samples increases, we gain more insight into the significance and function of each cytokine and the importance of cytokine networks. Previous studies that reported measurements of cytokine concentrations from serum or plasma in dogs with infectious, autoimmune, metabolic, endocrine, and neoplastic diseases yield an appreciation for the complexity of cytokine control and potential applications for cytokine measurements in the diagnosis, prognosis, and therapy of a variety of disease conditions. In this review, we highlight the benefits of multiplex cytokine analysis, summarize clinical and experimental reports that have used this technology in dogs, and discuss the strengths and limitations of data analysis for the interpretation of results in these studies. We describe how differences in technical acuity, data reporting tactics, statistical analysis, study population selection criteria, and cross-sectional experimental design methods may affect interpretation of results from this technology. We also suggest methods for analysis in future studies, such as reporting median fluorescence intensity values, analyzing the proportion of patients above population medians, and performing longitudinal studies. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum infections are especially severe in pregnant women because infected erythrocytes (IE) express VAR2CSA, a ligand that binds to placental trophoblasts, causing IE to accumulate in the placenta. Resulting inflammation and pathology increases a woman's risk of anemia, miscarriage, premature deliveries, and having low birthweight (LBW) babies. Antibodies (Ab) to VAR2CSA reduce placental parasitaemia and improve pregnancy outcomes. Currently, no single assay is able to predict if a woman has adequate immunity to prevent placental malaria (PM). This study measured Ab levels to 28 malarial antigens and used the data to develop statistical models for predicting if a woman has sufficient immunity to prevent PM. Methods: Archival plasma samples from 1377 women were screened in a bead-based multiplex assay for Ab to 17 VAR2CSA-associated antigens (full length VAR2CSA (FV2), DBL 1-6 of the FCR3, 3D7 and 7G8 lines, ID1-ID2a (FCR3 and 3D7) and 11 antigens that have been reported to be associated with immunity to P. falciparum (AMA-1, CSP, EBA-175, LSA1, MSP1, MSP2, MSP3, MSP11, Pf41, Pf70 and RESA)). Ab levels along with clinical variables (age, gravidity) were used in the following seven statistical approaches: logistic regression full model, logistic regression reduced model, recursive partitioning, random forests, linear discriminant analysis, quadratic discriminant analysis, and support vector machine. Results: The best and simplest model proved to be the logistic regression reduced model. AMA-1, MSP2, EBA-175, Pf41, and MSP11 were found to be the top five most important predictors for the PM status based on overall prediction performance. Conclusions: Not surprising, significant differences were observed between PM positive (PM+) and PM negative (PM-) groups for Ab levels to the majority of malaria antigens. Individually though, these malarial antigens did not achieve reasonably high performances in terms of predicting the PM status. Utilizing multiple antigens in predictive models considerably improved discrimination power compared to individual assays. Among seven different classifiers considered, the reduced logistic regression model produces the best overall predictive performance. [ABSTRACT FROM AUTHOR]

3D plasmonic colloidosomes are superior SERS sensors owing to their high sensitivity and excellent tolerance to laser misalignment. Herein, we incorporate plasmonic colloidosomes in a microfluidic channel for online SERS detection. Our method resolves the poor signal reproducibility and inter-sample contamination in the existing online SERS platforms. Our flow system offers rapid and continuous online detection of 20 samples in less than 5 min with excellent signal reproducibility. The isolated colloidosomes prevent cross-sample and channel contamination, allowing accurate quantification of samples over a concentration range of five orders of magnitude. Our system demonstrates high-resolution multiplex detection with fully preserved signal and Raman features of individual analytes in a mixture. High-throughput multi-assay analysis is performed, which highlights that our system is capable of rapid identification and quantification of a sequence of samples containing various analytes and concentrations. [ABSTRACT FROM AUTHOR]

Introduction: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. [ABSTRACT FROM PUBLISHER]

Advances in mass spectrometry, genome sequencing techniques, and bioinformatic strategies have accelerated the discovery of cancer-specific neoantigens. Tumors express multiple immunogenic neoantigens, and neoantigen-specific T cell receptors (TCRs) can be identified in peripheral blood's mononuclear cells in cancer patients. Therefore, individualized TCR-based therapies are a promising approach whereby multiple neoantigen-specific TCRs can be selected in each patient, potentially leading to a highly effective treatment for cancer patients. We developed three multiplex analytical assays to determine the quality attributes of the TCR-T cell drug product with a mixture of five engineered TCRs. The identity of each TCR was determined by two NGS-based methods, Illumina MiSeq and PacBio platforms. This approach not only confirms the expected TCR sequences but also differentiates them by their variable regions. The five individual TCR and total TCR knock-in efficiencies were measured by droplet digital PCR using specific reverse primers. A potency assay based on transfection of antigen-encoding-RNA was developed to assess the dose-dependent activation of T cells for each TCR by measuring the surface activation marker CD137 expression and cytokine secretion. This work provides new assays to characterize individualized TCR-T cell products and insights into quality attributes for the control strategy. [Display omitted] [ABSTRACT FROM AUTHOR]

Objectives: Antibody response on polysaccharide- and protein-based vaccines is useful to test B cell functionality. As only few studies have explored the value of studying immune response to both vaccines, we evaluated the clinical value of anti-polysaccharide and anti-protein Luminex-based multiplex assays in context of primary immunodeficiency (PID) diagnosis., Methods: A 10-plex Luminex-based assay detecting antibodies to ten pneumococcal polysaccharide (PnPS) serotypes [present in unconjugated Pneumovax, not in 13-valent pneumococcal conjugated vaccine (PCV)] and a 5-plex assay detecting antibodies to five protein antigens (present in DTap/Tdap) were clinically validated in healthy individuals (n=99) and in retrospective (n=399) and prospective (n=108) patient cohorts. Clinical features of individuals with impaired response to PnPS and/or proteins were compared to those with normal response., Results: Antigen-specific antibody thresholds were determined in healthy individuals. Individuals with impaired anti-PnPS responses and deficient immunoglobulin levels suffered more from autoimmune diseases and had lower B cell levels compared to individuals with impaired anti-PnPS response with normal immunoglobulin levels. Individuals with combined impaired response to PnPS and proteins showed more severe clinical manifestations compared to individuals with isolated impaired response to PnPS or proteins. Eight of the 11 individuals with severely impaired responses to both PnPS and proteins had common variable immunodeficiency. Evaluation of the anti-PnPS response to four serotypes not contained in 20-valent PCV was comparable to evaluation to ten serotypes not contained in 13-valent PCV., Conclusions: Multiplexed assessment of anti-PnPS and anti-protein responses combined with immunoglobulin quantification provides useful clinical information to support PID diagnosis., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

• This is a multiple comparison study of multiplex molecular platforms for the detection of 8 respiratory viruses from pediatric specimens. • Assays that were compared are BioFire® FilmArray® respiratory panel (FilmArray), Luminex NxTag® respiratory pathogen panel (NxTag RPP) and applied biosystems TaqMan array card (TAC). • Results from all the platforms were compared to singleplex rRT-PCRs (gold standard) targeting each of the 8 respiratory viruses. • All the three assays displayed high overall agreement (>90%) and high specificity (>90%) compared to the reference method. • Findings from our study provide confidence in the agreement and consistency of results obtained from different testing methods employed in a multi-site surveillance study. Acute viral respiratory infections are a major health burden in children worldwide. In recent years, rapid and sensitive multiplex nucleic acid amplification tests (NAATs) have replaced conventional methods for routine virus detection in the clinical laboratory. We compared BioFire® FilmArray® Respiratory Panel (FilmArray V1.7), Luminex NxTag® Respiratory Pathogen Panel (NxTag RPP) and Applied Biosystems TaqMan Array Card (TAC) for the detection of eight viruses in pediatric respiratory specimens. Results from the three platforms were analyzed with a single-plex real-time RT-PCR (rRT-PCR) assay for each virus. Of the 170/210 single-plex virus-positive samples, FilmArray detected a virus in 166 (97.6%), TAC in 163 (95.8%) and NxTag RPP in 160 (94.1%) samples. The Positive Percent Agreement (PPA) of FilmArray, NxTag RPP and TAC was highest for influenza B (100%, 100% and 95.2% respectively) and lowest for seasonal coronaviruses on both FilmArray (90.2%) and NxTag RPP (81.8%), and for parainfluenza viruses 1- 4 on TAC (84%). The Negative Percent Agreement (NPA) was lowest for rhinovirus/enterovirus (92.9%, 96.7% and 97.3%) on FilmArray, NxTag RPP and TAC respectively. NPA for all three platforms was highest (100%) for both parainfluenza viruses 1- 4 and influenza A and B, and 100% for human metapneumovirus with TAC as well. All three multiplex platforms displayed high overall agreement (>90%) and high NPA (>90%), while PPA was pathogen dependent and varied among platforms; high PPA (>90%) was observed for FilmArray for all eight viruses, TAC for six viruses and NxTag RPP for 4 viruses. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study was to investigate the percentage of such asymptomatic human bocavirus (HBoV) DNA-shedding patients with a sensitive commercial method. Materials & methods: Bronchoalveolar lavage fluids were analyzed with the xTag® respiratory viral panel (RVP) assay (Luminex, The Netherlands), the RespiFinder® SMART assay (PathoFinder, Maastricht, The Netherlands) and an in-house real-time PCR protocol. Results: While replacing the Luminex xTag RVP assay with the RespiFinder SMART assay for routine diagnostics, surprisingly, more than 50% of bronchoalveolar lavage fluid samples from asymptomatic patients previously tested with negative Luminex xTag RVP tested positive for HBoV. Conclusion: As the RespiFinder SMART assay has a higher sensitivity in the present study, this supports the theory that HBoV persists and is capable of shedding low copy numbers of viral genomes at very low numbers, even in asymptomatic people. [ABSTRACT FROM AUTHOR]

Abstract: Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation in DNA repair capacity for human health, and discusses the status of DNA repair assays as potential clinical tools for personalized prevention or treatment of disease. In particular, we highlight research showing that there are significant inter-individual variations in DNA repair capacity (DRC), and that measuring these differences provides important biological insight regarding disease susceptibility and cancer treatment efficacy. We emphasize work showing that it is important to measure repair capacity in multiple pathways, and that functional assays are required to fill a gap left by genome wide association studies, global gene expression and proteomics. Finally, we discuss research that will be needed to overcome barriers that currently limit the use of DNA repair assays in the clinic. [Copyright &y& Elsevier]

Background Similarly to the rest of the world, southern Africa's diverse chondrichthyan fauna is currently experiencing high fishing pressures from direct and non-direct fisheries to satisfy market demands for shark products such as fins and meat. In this study, the development of microsatellite markers through cross-species amplification of primer sets previously developed for closely related species is reported as an alternative approach to de novo marker development. This included the design of four microsatellite multiplex assays and their crossspecies utility in genetic diversity analysis of southern African elasmobranchs. As this study forms part of a larger project on the development of genetic resources for commercially important and endemic southern African species, Mustelus mustelus was used as a candidate species for testing these multiplex assays in down-stream applications. Results Thirty five microsatellite primer sets previously developed for five elasmobranch species were selected from literature for testing cross-species amplification in 16 elasmobranch species occurring in southern Africa. Cross-species amplification success rates ranged from 28.6%-71.4%. From the successfully amplified microsatellites, 22 loci were selected and evaluated for levels of polymorphism, and four multiplex assays comprising of the 22 microsatellites were successfully constructed, optimised and characterised in a panel of 87 Mustelus mustelus individuals. A total of 125 alleles were observed across all loci, with the number of alleles ranging from 3-12 alleles. Cross-species amplification of the four optimised multiplex assays was further tested on 11 commercially important and endemic southern African elasmobranch species. Percentage of polymorphism ranged from 31.8%-95.5% in these species with polymorphic information content decreasing exponentially with evolutionary distance from the source species. Conclusions Cross-species amplification of the 35 microsatellites proved to be a time- and cost-effective approach to marker development in elasmobranchs and enabled the construction of four novel multiplex assays for characterising genetic diversity in a number of southern African elasmobranch species. This study successfully demonstrated the usefulness of these markers in down-stream applications such as genetic diversity assessment and species identification which could potentially aid in a more integrative, multidisciplinary approach to management and conservation of commercially important cosmopolitan and endemic elasmobranch species occurring in southern Africa. [ABSTRACT FROM AUTHOR]

A fiber-optic based flow cytometry platform was designed to build a portable and robust instrument for space applications. At the core of the Microflow1 is a unique fiber-optic flow cell fitted to a fluidic system and fiber coupled to the source and detection channels. A Microflow1 engineering unit was first tested and benchmarked against a commercial flow cytometer as a reference in a standard laboratory environment. Testing in parabolic flight campaigns was performed to establish Microflow1's performance in weightlessness, before operating the new platform on the International Space Station. Microflow1 had comparable performances to commercial systems, and operated remarkably and robustly in weightlessness (microgravity). Microflow1 supported immunophenotyping as well as microbead-based multiplexed cytokine assays in the space environment and independently of gravity levels. Results presented here provide evidence that this fiber-optic cytometer technology is inherently compatible with the space environment with negligible compromise to analytical performance. Published by Wiley-Periodicals, Inc. © 2013 Government of Canada [ABSTRACT FROM AUTHOR]

Background As a part of the longitudinal Chronic Obstructive Pulmonary Disease (COPD) study, Subpopulations and Intermediate Outcome Measures in COPD study (SPIROMICS), blood samples are being collected from 3200 subjects with the goal of identifying blood biomarkers for sub-phenotyping patients and predicting disease progression. To determine the most reliable sample type for measuring specific blood analytes in the cohort, a pilot study was performed from a subset of 24 subjects comparing serum, Ethylenediaminetetraacetic acid (EDTA) plasma, and EDTA plasma with proteinase inhibitors (P100™). Methods 105 analytes, chosen for potential relevance to COPD, arranged in 12 multiplex and one simplex platform (Myriad-RBM) were evaluated in duplicate from the three sample types from 24 subjects. The reliability coefficient and the coefficient of variation (CV) were calculated. The performance of each analyte and mean analyte levels were evaluated across sample types. Results 20% of analytes were not consistently detectable in any sample type. Higher reliability and/or smaller CV were determined for 12 analytes in EDTA plasma compared to serum, and for 11 analytes in serum compared to EDTA plasma. While reliability measures were similar for EDTA plasma and P100 plasma for a majority of analytes, CV was modestly increased in P100 plasma for eight analytes. Each analyte within a multiplex produced independent measurement characteristics, complicating selection of sample type for individual multiplexes. Conclusions There were notable detectability and measurability differences between serum and plasma. Multiplexing may not be ideal if large reliability differences exist across analytes measured within the multiplex, especially if values differ based on sample type. For some analytes, the large CV should be considered during experimental design, and the use of duplicate and/or triplicate samples may be necessary. These results should prove useful for studies evaluating selection of samples for evaluation of potential blood biomarkers. [ABSTRACT FROM AUTHOR]

Elucidating how molecules bind to HSA is fundamental for predicting drug incompatibilities. Through combinatorial screening, we identified a novel fluorescent dye (BD140) with turn-on fluorescence emission and specific binding at HSA drug site 2. We further combined it with dansylamide to develop a fluorescent dye cocktail for high-throughput mapping of the interaction between therapeutics at HSA drug-binding sites. [ABSTRACT FROM AUTHOR]

Diagnostic testing has been improving the quality of cancer care. The dynamics of this field can be grasped through the application of innovation lifecycle models. Single testing, parallel testing and whole-genome sequencing are major technological evolutions. Given the increasing availability of biomarkers, the performance of single testing will be limited in the future, favoring the further implementation of parallel testing technologies. Whole-genome sequencing will lead to a further performance increase by introducing the era of genomic medicine. A broad adoption of presently available diagnostic technologies sets up the infrastructure for future technologies. The speed at which these technologies are introduced depends heavily on the regulatory and reimbursement environment, while their final diffusion is subject to pragmatic criteria such as user friendliness, perceived risk and perceived value added. [ABSTRACT FROM AUTHOR]

A GO‐to solution: A simple graphene oxide (GO)‐based assay to screen for selective inhibitors of a hepatitis C virus (HCV) helicase along with inhibitors of a severe acute respiratory syndrome coronavirus (SARS CoV) helicase was tested (see scheme). A single screen found five inhibitors highly selective for the HCV helicase orthologous to the SARS CoV helicase. Some of these hits were validated using the same GO‐based assay. [ABSTRACT FROM AUTHOR]

Molecular approaches to allergen standardization include the development of purified natural or recombinant allergen standards whose structural and allergenic properties have been validated, in tandem with certified immunoassays for allergen measurement. Purified allergens can be used individually or incorporated into multiple allergen standards. Multicenter international collaborative studies are required to validate candidate allergen standards and immunoassays, as a prelude to being approved by regulatory agencies. Mass spectrometry is a sophisticated and powerful proteomics tool that is being developed for allergen analysis. Recent results using pollen allergens show that mass spectrometry can identify and measure specific allergens in a complex mixture and can provide precise information of the variability of natural allergen extracts. In future, the combined use of immunoassays and mass spectrometry will provide complete standardization of allergenic products. Molecular standardization will form the basis of new allergy diagnostics and therapeutics, as well as assessment of environmental exposure, and will improve the quality of treatment options for allergic patients. [ABSTRACT FROM AUTHOR]

Foodborne illnesses caused by pathogenic bacteria represent a widespread and growing problem to public health, and there is an obvious need for rapid detection of food pathogens. Traditional culture-based techniques require tedious sample workup and are time-consuming. It is expected that new and more rapid methods can replace current techniques. To enable large scale screening procedures, new multiplex analytical formats are being developed, and these allow the detection and/or identification of more than one pathogen in a single analytical run, thus cutting assay times and costs. We review here recent advancements in the field of rapid multiplex analytical methods for foodborne pathogenic bacteria. A variety of strategies, such as multiplex polymerase chain reaction assays, microarray- or multichannel-based immunoassays, biosensors, and fingerprint-based approaches (such as mass spectrometry, electronic nose, or vibrational spectroscopic analysis of whole bacterial cells), have been explored. In addition, various technological solutions have been adopted to improve detectability and to eliminate interferences, although in most cases a brief pre-enrichment step is still required. This review also covers the progress, limitations and future challenges of these approaches and emphasizes the advantages of new separative techniques to selectively fractionate bacteria, thus increasing multiplexing capabilities and simplifying sample preparation procedures. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Pooled short-hairpin RNA (shRNA) library screening is a powerful tool for identifying a set of genes in biological pathways that require stable expression to produce a desired phenotype. Massive parallel sequencing of half-hairpins has proven highly variable and has not given satisfactory results concerning the relative abundance of different shRNAs before and after selection. Here, the authors describe a method for quantitative comparison of half-hairpins from pooled shRNAs in the mir30-based pGIPZ vector that is analyzed by massive parallel sequencing. Introducing a multiplexing code and refining the sample preparation scheme resulted in the predicted ability to detect twofold enrichments. These improvements should permit half-hairpin sequencing to analyze either dropout screens or selective pooled shRNA screens of limited stringency to analyze phenotypes not accessible in transient experiments. [ABSTRACT FROM PUBLISHER]

The benefits of probiotic supplementation to lactating mothers on human milk cytokines are inconclusive. Thus, we performed a comprehensive open-label pilot trial analysis of 27 human milk cytokines in lactating women with allergies (one to three months postpartum) to determine the effect of supplementation with a mixture of new probiotic strains. Participants voluntarily joined the probiotic (n = 41) or no supplementation control (n = 19) groups. The probiotic group took three probiotic tablets (Lactobacillus casei LC5, Bifidobacterium longum BG7, and Bacillus coagulans SANK70258) daily for one to three months postpartum. Milk samples were collected at one, two, and three months postpartum, and cytokine levels were measured using multiplex assays. The effects were analyzed using multivariate regression models. Eleven cytokines showed a positive rate of over 50% in the milk samples throughout testing in both groups. The positive rates of IL-1 receptor antagonist and IL-7 changed significantly with lactation progression in logistic regression models after adjusting for time and supplementation, whereas rates of other cytokines showed no significant differences. The lactational change patterns of IL-10 concentrations differed significantly between the two groups. A short-term supplementation of probiotics affects human milk cytokine levels in lactating women with a possible placebo effect still existing. Future placebo-controlled studies are needed to support these results, based on the estimated sample sizes in this study. [ABSTRACT FROM AUTHOR]