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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