Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA.
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.
Department of Biomedical Engineering, University of Michigan.
John P
JP
Frampton
Department of Biomedical Engineering, University of Michigan.
Joshua B
JB
White
Department of Macromolecular Science and Engineering, University of Michigan.
Arlyne B
AB
Simon
Department of Biomedical Engineering, University of Michigan.
Michael
M
Tsuei
Department of Pediatrics, Indiana University School of Medicine.
Sophie
S
Paczesny
1] Department of Biomedical Engineering, University of Michigan [2] Department of Macromolecular Science and Engineering, University of Michigan.
Shuichi
S
Takayama
D000906:Antibodies / Q000097:blood* / Q000276:immunology*; D015415:Biomarkers / Q000097:blood; D016026:Bone Marrow Transplantation / Q000379:methods; D003429:Cross Reactions / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006086:Graft vs Host Disease / Q000097:blood / Q000276:immunology; D006801:Humans
D000906:Antibodies; D015415:Biomarkers