Journal: Toxins

Article Title: Highly Sensitive Suspension Immunoassay for Multiplex Detection, Differentiation, and Quantification of Eight Staphylococcus aureus Enterotoxins (SEA to SEI)

doi: 10.3390/toxins17060265

Figure Lengend Snippet: Detection of SEs by sandwich ELISAs and multiplex SIA. The sandwich ELISAs (top panel) were set up using specific combinations of immobilised capture mAbs, SEA388 + SEA2353 (for SEA), S1001 (SEB), SEC371 (SEC), SED1280 (SED), SEE33 (SEE), SEG5 (SEG), SEH1236 (SEH), and SEI467 (SEI). Each of these capture mAbs was paired with a corresponding detection mAb, SEA165 (SEA), S419 (SEB), SEC290 (SEC), SED333 (SED), SEE1524 (SEE), SEG158 (SEG), SEH449 (SEH), and SEI92 (SEI). The assays were performed in 96-well microtiter plates. For the multiplex SIA, the assay was adapted to a bead-based format (bottom panel). In this format, paramagnetic beads were coated with mAbs: SEA388 + SEA2353, S1851, SEC371, SED1280, SEE1524, SEG5, SEH449, and SEI242 (from left to right, specific for SEA to SEI) on different bead sets. Detection was achieved using a mixture of biotinylated detection mAbs: SEA165, S419, SEC290, SED9, SEE33, SEG158, SEH1236, and SEI92 (left to right, specific for SEA to SEI), allowing for simultaneous detection of multiple SE targets. Detection was performed after incubation of capture mAbs with serial dilutions of SE antigens—SEA (black), SEB (orange), SEC1 (light blue), SED (green), SEE (yellow), SEG (dark blue), SEH (brown), and SEI (purple)—in both assay formats. The ELISAs used single biotinylated mAbs in combination with streptavidin–horseradish peroxidase for detection, while the multiplex SIA used a mixture of biotinylated mAbs and streptavidin–phycoerythrin. The results were recorded as A [450–620nm] , corresponding to the absorbance measured at 450 nm minus the one at 620 nm, for the individual ELISAs and as the MFI (median fluorescent intensity) for the multiplex SIA. The results were modelled using a semi-logarithmic four-parameter fit with log-transformed concentration values ( x -axis). Data for the specific target antigens in their respective ELISA or on their specific beads were collected from five independent experiments, each measured in duplicate ( n = 5). In addition, to assess potential cross-reactivity, each specific SE antigen was tested on the other respective seven ELISAs or bead sets in three independent experiments ( n = 3).

Article Snippet: Following a 30 min incubation period with phycoerythrin-coupled streptavidin (SA-PE; Agilent, Santa Clara, CA, USA) in 1% ( w / v ) BSA in PBS, the plate was washed (3 × 300 μL) and the beads were resuspended in 125 μL PBS with 0.05% ( w / v ) NaN 3 .

Techniques: Multiplex Assay, Incubation, Transformation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Toxins

Article Title: Highly Sensitive Suspension Immunoassay for Multiplex Detection, Differentiation, and Quantification of Eight Staphylococcus aureus Enterotoxins (SEA to SEI)

doi: 10.3390/toxins17060265

Figure Lengend Snippet: Multiplex SIA results for the detection of clustered native toxins produced by 145 bacterial strains. After immobilising capture mAbs on individual magnetic microbeads for the specific detection of SEA to SEI (mAbs SEA388 + SEA2353, S1851, SEC371, SED1280, SEE1524, SEG5, SEH449, and SEI242), a total of 145 culture supernatants were incubated as 1:10 diluted samples with the antibody-coated microbeads, washed, and detected by a mixture of biotinylated detection mAbs (SEA165, S419, SEC290, SED9, SEE33, SEG158, SEH1236, and SEI92) followed by streptavidin–phycoerythrin. The results based on ROC analysis (pROC package, version 1.18.0) using R (version 4.1.3) were compared to filtered sea - to sei -genic strains based on whole genome sequencing (WGS) data. The cluster size (number of strains) is given as n. The presence of toxins in the genomic sequence data is indicated by fields with a grey background. The colour of the circles denotes the correctness of the multiplex results in relation to the WGS data. Correctly identified SE protein results are shown as either green (true positive) or blue (true negative), while incorrect results are shown as either yellow (false positive) or orange (false negative). The thickness of the circle lines correlates positively with the cluster size.

Article Snippet: Following a 30 min incubation period with phycoerythrin-coupled streptavidin (SA-PE; Agilent, Santa Clara, CA, USA) in 1% ( w / v ) BSA in PBS, the plate was washed (3 × 300 μL) and the beads were resuspended in 125 μL PBS with 0.05% ( w / v ) NaN 3 .

Techniques: Multiplex Assay, Produced, Incubation, Sequencing

Journal: Toxins

Article Title: Highly Sensitive Suspension Immunoassay for Multiplex Detection, Differentiation, and Quantification of Eight Staphylococcus aureus Enterotoxins (SEA to SEI)

doi: 10.3390/toxins17060265

Figure Lengend Snippet: Performance parameters of the multiplex SIA for the detection of native toxins in bacterial culture supernatants. After immobilisation of the capture mAbs on paramagnetic microbeads, a total of 145 culture supernatants were diluted in a ratio of 1:10, incubated, and detected with a mixture of the detection mAbs directed against SEA to SEI (see ) and streptavidin–phycoerythrin. The results from three independent experiments ( n = 3), analysed by ROC analysis (pROC package, version 1.18.0) using R (version 4.1.3), were compared with whole genome sequencing data of the strains and calculated as performance metrics. The data are presented with median values (dots) and confidence intervals (bars) for each SE detected in the multiplex SIA, using three colours to represent the performance metrics: accuracy (black), sensitivity (orange), and specificity (blue).

Article Snippet: Following a 30 min incubation period with phycoerythrin-coupled streptavidin (SA-PE; Agilent, Santa Clara, CA, USA) in 1% ( w / v ) BSA in PBS, the plate was washed (3 × 300 μL) and the beads were resuspended in 125 μL PBS with 0.05% ( w / v ) NaN 3 .

Techniques: Multiplex Assay, Incubation, Sequencing

Journal: Toxins

Article Title: Highly Sensitive Suspension Immunoassay for Multiplex Detection, Differentiation, and Quantification of Eight Staphylococcus aureus Enterotoxins (SEA to SEI)

doi: 10.3390/toxins17060265

Figure Lengend Snippet: Overview of the estimated toxin concentration in all supernatants obtained from sea- to sei -genic strains measured by multiplex SIA. After immobilisation of the capture mAbs on paramagnetic microbeads, a set of 145 culture supernatants was incubated as samples. SE detection was performed using a mixture of biotinylated detection mAbs, followed by the addition of streptavidin–phycoerythrin. In this experiment ( n = 2), the results were extrapolated to the toxin dilution curve, with each concentration per target and supernatant from the target-encoding strain represented as a black dot. The number of black dots reflects the number of enterotoxigenic strains for each specific target SE; thus, only true positives and false negatives are shown. The mean concentration values across all target se -genic strains are depicted as a bar, with the standard deviation included. For an overview of the estimated toxin concentration in the 145 strains depicted over the different se variants, please see .

Article Snippet: Following a 30 min incubation period with phycoerythrin-coupled streptavidin (SA-PE; Agilent, Santa Clara, CA, USA) in 1% ( w / v ) BSA in PBS, the plate was washed (3 × 300 μL) and the beads were resuspended in 125 μL PBS with 0.05% ( w / v ) NaN 3 .

Techniques: Concentration Assay, Multiplex Assay, Incubation, Standard Deviation

Journal: Toxins

Article Title: Highly Sensitive Suspension Immunoassay for Multiplex Detection, Differentiation, and Quantification of Eight Staphylococcus aureus Enterotoxins (SEA to SEI)

doi: 10.3390/toxins17060265

Figure Lengend Snippet: The recovery of SEA to SEI in undiluted food extracts by multiplex SIA. Magnetic microspheres were coated with capture mAbs directed against SEA to SEI on different bead sets. Undiluted food extracts were spiked with lower (1 × EC 50 ) or higher (10 × EC 50 ) concentrations of a toxin mixture containing SEA to SEI. Sample detection was conducted using a combination of SEA- to SEI-specific biotinylated detection mAbs, followed by the application of streptavidin–phycoerythrin. The measured values of the target antigens in the extracts and in buffer were converted into toxin concentrations. The efficiency with which a target antigen was detected in an extract, measured in five biological replicates compared to its detection in buffer, is shown here as recovery rate in per cent (for details see ), indicated with a scale from yellow for high recovery and dark blue for low recovery.

Article Snippet: Following a 30 min incubation period with phycoerythrin-coupled streptavidin (SA-PE; Agilent, Santa Clara, CA, USA) in 1% ( w / v ) BSA in PBS, the plate was washed (3 × 300 μL) and the beads were resuspended in 125 μL PBS with 0.05% ( w / v ) NaN 3 .

Techniques: Multiplex Assay