Aqueous Two-Phase System Rehydration of Antibody-Polymer Microarrays Enables Convenient Compartmentalized Multiplex Immunoassays.
Multiplex immunoassays are rapidly increasing in popularity due to the offered advantages of increased throughput and decreased sample volume requirements. However, a major weakness inherent to multiplex enzyme-linked immunosorbent assays (ELISA) is generation of false signals through reagent-driven cross-talk. Typically, multiplex platforms necessitate bath application of antibody cocktails, increasing probability of nonspecific antibody binding, especially when multiplexing large numbers of analytes. Aqueous two-phase systems (ATPS) exploiting the phase-separating polymers poly(ethylene) glycol (PEG) and dextran (DEX) have been used to compartmentalize antibodies and prevent cross-talk in multliplex, plate-based ELISA. However, the resulting protocol is tedious and lengthy, and requires too many user steps to be practical for widespread use. Here, we report an improved, user-friendly, cross-talk-free multiplex ELISA method in which dehydrated arrays of colocalized capture and detection antibodies in DEX are prepared on multiwell plates. Addition of a PEG-based sample buffer rehydrates antibody/DEX droplets for analysis. In this report, we demonstrate rehydrated ATPS components for multiplex ELISA retain the ability to compartmentalize antibodies and prevent cross-talk, while analytes in sample buffer partition into rehydrated DEX droplets for analysis. Utility of this method was demonstrated through successful quantitative analysis of five inflammatory cytokines in lipopolysaccharide-stimulated ThP-1 cell culture supernatant.
PHASIQ, Inc. , Ann Arbor, Michigan 48109, United States.
Lisa
L
Eiden
Department of Biomedical Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States.
Cameron
C
Yamanishi
Department of Biomedical Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States.|Department of Macromolecular Science and Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States.
Shuichi
S
Takayama
PHASIQ, Inc. , Ann Arbor, Michigan 48109, United States.
John F
JF
Dishinger
D000906:Antibodies / Q000032:analysis*; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis / Q000096:biosynthesis; D003911:Dextrans / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005440:Fluid Therapy; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000737:chemistry*; D014867:Water / Q000737:chemistry
D000906:Antibodies; D016207:Cytokines; D003911:Dextrans; D008070:Lipopolysaccharides; D014867:Water; D011092:Polyethylene Glycols