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Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA.

Frampton JP, White JB, Simon AB, Tsuei M, Paczesny S, Takayama S - Sci Rep (2014)

Bottom Line: This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients.The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced.Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Michigan.

ABSTRACT
Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

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Related in: MedlinePlus

DEX droplets assume dome shapes that change very little in size and shape over the course of the incubation period.(a) Antibodies are retained in the DEX domes over this period as indicated by the overlap between FITC-DEX and PE-IgG. Scale bar = 1 mm. (b) Biotin-labeled ELISA detection antibodies partition favorable to the DEX phase. Partition coefficients were measured by blotting detection antibody fractions from PEG and DEX on PVDF membranes and detecting the antibody levels by way of streptavidin-HRP chemiluminescence. Partitioning can be further improved by modifying ATPS formulations. (c) Partial overlap of the capture and detection antibodies results in a cat eye shape that can only be produced if antibodies are well retained in the DEX droplet. (d) Bath application of detection antibodies produced a circular signal area.
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f2: DEX droplets assume dome shapes that change very little in size and shape over the course of the incubation period.(a) Antibodies are retained in the DEX domes over this period as indicated by the overlap between FITC-DEX and PE-IgG. Scale bar = 1 mm. (b) Biotin-labeled ELISA detection antibodies partition favorable to the DEX phase. Partition coefficients were measured by blotting detection antibody fractions from PEG and DEX on PVDF membranes and detecting the antibody levels by way of streptavidin-HRP chemiluminescence. Partitioning can be further improved by modifying ATPS formulations. (c) Partial overlap of the capture and detection antibodies results in a cat eye shape that can only be produced if antibodies are well retained in the DEX droplet. (d) Bath application of detection antibodies produced a circular signal area.

Mentions: ATPS-ELISA is performed using the same steps as conventional sandwich ELISA, except that ATPS-ELISA uses detection antibody solutions that are deposited in DEX droplets over the capture antibody spots (Figure 1 a and b) by micropipetting. This prevents cross-reactions between unwanted pairs of polyclonal capture and detection antibodies, as well as from non-target antigen recognition by detection antibodies. The capture antibodies and detection antibodies can be easily aligned using polystyrene plates with embossed features (Figure S1), although it is also possible to perform the assay using planar polystyrene substrates, as shown in Figures 1 and 2. Figure 1 c shows the effectiveness of ATPS-ELISA at eliminating antibody cross-reactions. We intentionally spotted polyclonal antibodies that are more prone to cross-react. One spot contained an immobilized anti-human ST2 capture antibody, while the adjacent spot contained an immobilized anti-goat antibody that recognized the detection antibody (a goat anti-human ST2 antibody). After incubating with a solution containing ST2, the goat detection antibody was either deposited above the mouse capture antibody-antigen complex in DEX using an ATPS or bath applied in the conventional manner without using ATPS (Figure 1 c, d). There was no cross-reactive signal for the ATPS format (Figure 1 c), demonstrating that the detection antibodies remained confined to DEX and were unable to diffuse away to react with the anti-goat antibody spot. On the other hand, conventional bath application allowed detection antibodies to freely circulate, resulting in strong signals at both the appropriate capture antibody spot and the spot containing the anti-goat antibody (Figure 1 d). This illustrates the possibility of false-positive readouts that may lead to misinterpretation of the biomarker panel when inappropriate combinations of detection antibodies are used in conventional multiplex systems.


Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA.

Frampton JP, White JB, Simon AB, Tsuei M, Paczesny S, Takayama S - Sci Rep (2014)

DEX droplets assume dome shapes that change very little in size and shape over the course of the incubation period.(a) Antibodies are retained in the DEX domes over this period as indicated by the overlap between FITC-DEX and PE-IgG. Scale bar = 1 mm. (b) Biotin-labeled ELISA detection antibodies partition favorable to the DEX phase. Partition coefficients were measured by blotting detection antibody fractions from PEG and DEX on PVDF membranes and detecting the antibody levels by way of streptavidin-HRP chemiluminescence. Partitioning can be further improved by modifying ATPS formulations. (c) Partial overlap of the capture and detection antibodies results in a cat eye shape that can only be produced if antibodies are well retained in the DEX droplet. (d) Bath application of detection antibodies produced a circular signal area.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4007081&req=5

f2: DEX droplets assume dome shapes that change very little in size and shape over the course of the incubation period.(a) Antibodies are retained in the DEX domes over this period as indicated by the overlap between FITC-DEX and PE-IgG. Scale bar = 1 mm. (b) Biotin-labeled ELISA detection antibodies partition favorable to the DEX phase. Partition coefficients were measured by blotting detection antibody fractions from PEG and DEX on PVDF membranes and detecting the antibody levels by way of streptavidin-HRP chemiluminescence. Partitioning can be further improved by modifying ATPS formulations. (c) Partial overlap of the capture and detection antibodies results in a cat eye shape that can only be produced if antibodies are well retained in the DEX droplet. (d) Bath application of detection antibodies produced a circular signal area.
Mentions: ATPS-ELISA is performed using the same steps as conventional sandwich ELISA, except that ATPS-ELISA uses detection antibody solutions that are deposited in DEX droplets over the capture antibody spots (Figure 1 a and b) by micropipetting. This prevents cross-reactions between unwanted pairs of polyclonal capture and detection antibodies, as well as from non-target antigen recognition by detection antibodies. The capture antibodies and detection antibodies can be easily aligned using polystyrene plates with embossed features (Figure S1), although it is also possible to perform the assay using planar polystyrene substrates, as shown in Figures 1 and 2. Figure 1 c shows the effectiveness of ATPS-ELISA at eliminating antibody cross-reactions. We intentionally spotted polyclonal antibodies that are more prone to cross-react. One spot contained an immobilized anti-human ST2 capture antibody, while the adjacent spot contained an immobilized anti-goat antibody that recognized the detection antibody (a goat anti-human ST2 antibody). After incubating with a solution containing ST2, the goat detection antibody was either deposited above the mouse capture antibody-antigen complex in DEX using an ATPS or bath applied in the conventional manner without using ATPS (Figure 1 c, d). There was no cross-reactive signal for the ATPS format (Figure 1 c), demonstrating that the detection antibodies remained confined to DEX and were unable to diffuse away to react with the anti-goat antibody spot. On the other hand, conventional bath application allowed detection antibodies to freely circulate, resulting in strong signals at both the appropriate capture antibody spot and the spot containing the anti-goat antibody (Figure 1 d). This illustrates the possibility of false-positive readouts that may lead to misinterpretation of the biomarker panel when inappropriate combinations of detection antibodies are used in conventional multiplex systems.

Bottom Line: This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients.The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced.Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Michigan.

ABSTRACT
Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

Show MeSH
Related in: MedlinePlus