Fig 1.
Coating of microcapsules and beads with proteins or nucleic acids: The carboxylic acid groups in the outmost layer of the microcapsules were surface-activated by EDC/sulfo-NHS. Then, either protein A was attached onto the activated surface for oriented binding of antibodies (A), or streptavidin was immobilized for the binding of biotinylated oligonucleotides (B). The coating of commercially available beads was performed in the same way. Drawings are not to scale.
Fig 2.
Comparison of 6 μm microcapsules and 2.35 μm PS beads for binding of antibody and for the detection of a protein analyte: (A+B) Detection of the capture antibody, BBM.1, on microcapsules and beads with fluorescently labeled goat anti-mouse antibody (GαM-AF488) by flow cytometry. (A) shows a representative experiment, (B) the average of two experiments with standard deviations (SD). (C) Schematics for the detection of human beta-2 microglobulin (hβ2m). The capture antibody, BBM.1, is immobilized on the protein A-coated microcapsules/PS beads. BBM.1 antibody binds specifically to hβ2m, which is sandwiched by the polyclonal rabbit anti-hβ2M (Rαhβ2M) antibody. The sandwich is then detected by adding AF488 labeled goat anti-rabbit (GαR-AF488) antibody. (D) Detection of hβ2m in PBS. Dose-response curves for the assay performed as in (C) with microcapsules or PS beads. MFI values are normalized to the maximum values. Error bars are SD (n = 3). Invisible error bars are smaller than the size of the marker. (E) Control samples of hβ2m plotted as histograms. Experiments were performed as in (C). Samples with analyte (105 pg mL-1 for microcapsules and 106 pg mL-1 for PS beads) were used as positive control and for normalization, which was done individually for microcapsules and PS beads. Error bars are SD (n = 3).
Table 1.
Analytical performance of hβ2m and oligonucleotide detection using microcapsule and PS beads: Best fit values were obtained with a four-parameter fit equation.
Limit of blank (LoB), limit of detection (LoD) and limit of quantification (LoQ) were determined as described in the materials and methods.
Fig 3.
Comparison of microcapsules and PS beads for the detection of nucleic acids: (A) Schematics. The anchor, Biotin-Oligo2, is attached to streptavidin-coated microcapsules or PS beads andhybridizes specifically to its complementary sequence on the analyte and on Oligo3, which in turn hybridizes to its complementary sequence on the detector, Oligo4-FITC. (B) Analyte dose-response of the assay for streptavidin-coated microcapsules and PS beads measured by flow cytometry. MFI values were normalized by the maximum value. The error bars are SD (n = 3). Invisible error bars are smaller than the size of the marker. (C) Background controls for the assay in (B). MFI values were normalized to the maximum value. The error bars indicate the SD (n = 3). (D) Hybridization scheme of the three oligonucleotides used in the current assay. Biotin-Oligo2 hybridizes to the 17 complementary nucleotide bases (brown) of analyte Oligo3, and the remaining free 17 bases of the analyte hybridize to the detector Oligo4-FITC (green).
Fig 4.
Comparison of microcapsule and microplate assays in the detection of the BBM.1 antibody in hybridoma supernatant: (A-C) Analyte dose-response curves for the BBM.1 antibody in PBS (A), RPMI (B), and dilutions of hybridoma supernatant (C). Microcapsules or plates were coated with protein A. After binding of the analyte, samples were incubated with detector antibody GαM-AF488 and measured either by flow cytometry (microcapsules) or in a plate reader (microplates). LoDs are: in (A) 26 ng mL-1 for microcapsules and 24 ng mL-1 for plates; in (B) 7 ng mL-1 for microcapsules and 54 ng mL-1 for plates; in (C) 2.8 nL mL-1 for microcapsules and 158 nL mL-1 for plates. See Table 2 for complete data. (D-F) Background controls and comparisons for the curves above. For (D) and (E), 'Max analyte' is the largest amount of analyte used in the assay above (20 μg mL-1 for microcapsules and 10 μg mL-1 for the microplate). For (F), 'Max Hyb.' is the largest volume of hybridoma used in the assay above (2000 μL for microcapsules and 100 μL for microplate). For (E) and (F), 'Max. analyte (PBS)' is 30 μg μL-1 in PBS. MFI values were normalized by the highest value (A-C), the 'Max analyte' value (D-E), and the 'Max Hyb.' value (F). Error bars are the SD (n = 3). Invisible error bars are smaller than the size of the marker.
Table 2.
Analytical performance of BBM.1 detection using microcapsule and microplate for BBM.1 hybridoma: Best fit values were obtained with a four-parameter fit equation.
LoB, LoD, and LoQ were determined as described in the materials and methods.