Aqueous Two-Phase System Patterning for Crosstalk-free Multiplexed Detection of Plasma Biomarkers.

[摘要] Highly sensitive and robust protein assay platforms are urgently needed to improve diagnosis and inform therapeutic decisions in many disease areas. However, current bioassays for multiplexed protein biomarker detection are plagued by nonspecific antibody cross-reactions, which often lead to false positive detection and misdiagnoses. The work presented in this dissertation fills this key technological gap in protein assay development, and describes two innovative multiplexed bioassays that completely eliminate antibody cross-reactions. First, we introduce a multiplexed bead-based assay that not only prevents antibody cross-reactions, but also does not require any wash steps. We circumvented the antibody crosstalk problem by stably co-localizing antibody-conjugated beads in droplets of immiscible aqueous polymer solutions. We demonstrated this assay’s clinical utility by measuring the levels of proinflammatory biomarkers (chemokine ligand 9, chemokine ligand 10, interleukin-6, and interleuikin-8) in plasma samples from from 88 bone marrow transplant (BMT) patients. Our assay accurately discriminated BMT patients afflicted with and without chronic graft-versus-host disease (GVHD) patients within 2 hours.Second, we describe a multiplexed ELISA that eradicates antibody cross-reactions by confining and spatially patterning detection antibodies within droplets of phase-separating polymers. We used this crosstalk-free assay to measure a panel of four acute GVHD biomarkers (tumor necrosis factor-alpha, hepatocyte growth factor, elafin, and ST2) in plasma samples from 81 BMT patients. Not only does this assay eliminate antibody crosstalk, but it also reduces antibody costs by 100-fold because assays only require 0.5-microliters detection antibody volume instead of 50-microliters required by conventional ELISA.Notably, both crosstalk-free multiplexed platforms can be read using commercially available plate readers, indicating that these assay platforms can be valuable tools for diagnosis complex diseases like GVHD.

[发布机构] University of Michigan

[效力级别] Biomarkers

[关键词] Aqueous Two-phase Systems;Biomarkers;ELISA;AlphaLISA;Antibody Crosstalk;Multiplex Immunoassay;Biomedical Engineering;Engineering;Science;Macromolecular Science & Engineering