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

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.

Recent developments in multiplexing techniques for immunohistochemistry

“…More recently, ATPS has been used to generate multiplex antibody-based assays [58,59]. Importantly, many antibodies demonstrate strong partitioning to the dextran phase in an ATPS composed of polyethylene glycol and dextran (K≪1).…”

Aqueous two-phase systems enable multiplexing of homogeneous immunoassays

“…To mitigate risks of cross-reactions, tedious systematic evaluations of nonspecific interactions between each antibody and each measured protein must be performed to validate these assays 7–10 . Alternatively, matched capture and detection antibody pairs can also be co-localized 11,12 . Multiplexing AlphaLISA™ is even more challenging because single-color signals, generated by homogeneously distributed antibody-bead reagents, cannot be spatially localized within one well or spectrally resolved using conventional methods.…”

Liquid-in-liquid antibody confinement provides new possibilities for multiplexed diagnostics

“…In terms of general assay performance, this new multiplex assay platform is similar to standard single biomarker ELISA. Comparison data for limit of detection and linear dynamic range are reported in two recent publications that describe the antibody confinement technology [3,5].…”

“…In a recent report that measured plasma biomarkers by ELISA for assessment of therapy-resistant GVHD [6], an average receiver operating characteristic area under the curve (AUC; which is a measure of the sensitivity and specificity of the test for various biomarker cutoff concentrations) of 0.78 was obtained for a set of 12 individual GVHD biomarkers. The polymer-based ELISA system yielded a slightly higher average receiver operating characteristic AUC of 0.85 for a set of four individual GVHD biomarkers using a smaller set of clinical samples [5].…”

“…To address this limitation of multiplex immunoassays, an array-based immunoassay has recently been developed that eliminates the possibility of cross-reactions among antibodies in a multi-biomarker panel by confining the antibodies at specific sites on an assay plate [3][4][5]. Antibody confinement is enabled using polymer solutions that phase-separate from each other, much like oil droplets in water.…”