Abstract
Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity.
Highlights
Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins
We proved ambient analyte conditions for suspension bead arrays by repeatedly probing the same sample for the same analyte
A model assay involving an antibiotin antibody as capture molecule and a biotin-PE analyte was used to assess the validity of ambient analyte conditions in suspension bead arrays
Summary
Oliver Poetz‡§, Tanja Henzler¶, Michael Hartmann‡ʈ, Cornelia Kazmaier‡, Markus F. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibodycoated, magnetic suspension bead arrays. Either planar or bead array-based sandwich immunoassays can be used to analyze the quantity and activation state of signaling molecules in multiplex, enabling the systematic profiling of protein abundance and post-translational modifications [3,4,5,6] in hundreds of samples. A mixture of detection antibodies, one specific for the phosphorylation site and one specific for the nonmodified site of the protein, would bind simultaneously to the two different epitopes, and assay signals could not be further deconvoluted by the spatial or color code of the array. The second sandwich immunoassay setup for the analysis of protein phosphorylation applies a phosphorylation state-specific capture antibody and a protein-specific detection antibody.
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