Dataset Information

Beadplexr: reproducible and automated analysis of multiplex bead assays.

ABSTRACT: Multiplex bead assays are an extension of the commonly used sandwich ELISA. The advantage over ELISA is that they make simultaneous evaluation of several analytes possible. Several commercial assay systems, where the beads are acquired on a standard flow cytometer, exist. These assay systems come with their own software tool for analysis and evaluation of the concentration of the analyzed analytes. However, these tools are either tied to particular commercial software or impose other limitations to their licenses, such as the number of events which can be analyzed. In addition, all these solutions are 'point and click' which potentially obscures the steps taken in the analysis. Here we present beadplexer, an open-source R-package for the reproducible analysis of multiplex bead assay data. The package makes it possible to automatically identify bead clusters, and provides functionality to easily fit a standard curve and calculate the concentrations of the analyzed analytes. beadplexer is available from CRAN and from https://gitlab.com/ustervbo/beadplexr.

SUBMITTER: Stervbo U

PROVIDER: S-EPMC6241392 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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beadplexr: reproducible and automated analysis of multiplex bead assays.

Stervbo Ulrik U Westhoff Timm H TH Babel Nina N

PeerJ 20181116

Multiplex bead assays are an extension of the commonly used sandwich ELISA. The advantage over ELISA is that they make simultaneous evaluation of several analytes possible. Several commercial assay systems, where the beads are acquired on a standard flow cytometer, exist. These assay systems come with their own software tool for analysis and evaluation of the concentration of the analyzed analytes. However, these tools are either tied to particular commercial software or impose other limitations ...[more]

PMID: 30479885

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Project description:Serological assays for SARS-CoV-2 antibodies must be validated for performance with a large panel of clinical specimens. Most existing assays utilize a single antigen target and may be subject to reduced diagnostic specificity. This study evaluated a multiplex assay that detects antibodies to three SARS-CoV-2 targets. Human serum specimens (n = 323) with known previous SARS-CoV-2 exposure status were tested on a commercially available multiplex bead assay (MBA) measuring IgG to SARS-CoV-2 spike protein receptor-binding domain (RBD), nucleocapsid protein (NP), and RBD/NP fusion antigens. Assay performance was evaluated against reverse transcriptase PCR (RT-PCR) results and also compared with test results for two single-target commercial assays. The MBA had a diagnostic sensitivity of 89.8% and a specificity of 100%, with serum collection at >28 days following COVID-19 symptom onset showing the highest seropositivity rates (sensitivity: 94.7%). The MBA performed comparably to single-target assays with the ability to detect IgG against specific antigen targets, with 19 (5.9%) discrepant specimens compared to the NP IgG assay and 12 (3.7%) compared to the S1 RBD IgG assay (kappa coefficients 0.92 and 0.88 compared to NP IgG and S1 RBD IgG assays, respectively. These findings highlight inherent advantages of using a SARS-CoV-2 serological test with multiple antigen targets; specifically, the ability to detect IgG against RBD and NP antigens simultaneously. In particular, the 100.0% diagnostic specificity exhibited by the MBA in this study is important for its implementation in populations with low SARS-CoV-2 seroprevalence or where background antibody reactivity to SARS-CoV-2 antigens has been detected. IMPORTANCE Reporting of SARS-CoV-2 infections through nucleic acid or antigen based diagnostic tests severely underestimates the true burden of exposure in a population. Serological data assaying for antibodies against SARS-CoV-2 antigens offers an alternative source of data to estimate population exposure, but most current immunoassays only include a single target for antibody detection. This report outlines a direct comparison of a multiplex bead assay to two other commercial single-target assays in their ability to detect IgG against SARS-CoV-2 antigens. Against a well-defined panel of 323 serum specimens, diagnostic sensitivity and specificity were very high for the multiplex assay, with strong agreement in IgG detection for single targets compared to the single-target assays. Collection of more data for individual- and population-level seroprofiles allows further investigation into more accurate exposure estimates and research into the determinants of infection and convalescent responses.

Project description:BackgroundFew bead-based multiplex assays have been described that detect antibodies against the protozoan parasite Toxoplasma gondii in large-scale seroepidemiological surveys. Moreover, each multiplex assay has specific variations or limitations, such as the use of truncated or fusion proteins as antigens, potentially masking important epitopes. Consequently, such an assay must be developed by interested groups as none is commercially available.ResultsWe report the bacterial expression and use of N-terminal fusion-free, soluble, in vivo biotinylated recombinant surface antigens SAG1 and SAG2A for the detection of anti-T. gondii IgG antibodies. The expression system relies on three compatible plasmids. An expression construct produces a fusion of maltose-binding protein with SAG1 (or SAG2A), separated by a TEV protease cleavage site, followed by a peptide sequence recognized by E. coli biotin ligase BirA (AviTag), and a terminal six histidine tag for affinity purification. TEV protease and BirA are encoded on a second plasmid, and their expression leads to proteolytic cleavage of the fusion protein and a single biotinylated lysine within the AviTag by BirA. Correct folding of the parasite proteins is dependent on proper disulfide bonding, which is facilitated by a sulfhydryl oxidase and a protein disulfide isomerase, encoded on the third plasmid. The C-terminal biotinylation allowed the oriented, reproducible coupling of the purified surface antigens to magnetic Luminex beads, requiring only minute amounts of protein per determination. We showed that an N-terminal fusion partner such as maltose-binding protein negatively influenced antibody binding, confirming that access to SAG1's N-terminal epitopes is important for antibody recognition. We validated our bead-based multiplex assay with human sera previously tested with commercial diagnostic assays and found concordance of 98-100% regarding both, sensitivity and specificity, even when only biotinylated SAG1 was used as antigen.ConclusionsOur recombinant in vivo-biotinylated T. gondii antigens offer distinct advantages compared to previously described proteins used in multiplex serological assays for T. gondii. They offer a cheap, specific and sensitive alternative to either parasite lysates or eukaryotic-cell expressed SAG1/SAG2A for BBMA and other formats. The described general expression strategy can also be used for other antigens where oriented immobilization is key for sensitive recognition by antibodies and ligands.

Dataset Information

Beadplexr: reproducible and automated analysis of multiplex bead assays.

Publications

beadplexr: reproducible and automated analysis of multiplex bead assays.

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