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. Author manuscript; available in PMC: 2014 Dec 31.
Published in final edited form as: J Immunol Methods. 2013 Oct 24;0:10.1016/j.jim.2013.09.013. doi: 10.1016/j.jim.2013.09.013

Figure 4. Development of a Hybrid Multiplex Assay for Autoantibodies (TAAs) and Circulating Proteins.

Figure 4

A hybrid VeraCode™ Bead™ assay for multiplexed detection of serum autoantibodies to the p53 TAA (antigen-antibody assay format) as well as detection of the circulating serum proteins CEA and GDF15 (sandwich immunoassay format) was developed. (A.) To verify the sandwich immunoassay portion of the assay could be multiplexed, recombinant CEA or GDF15 standard proteins were spiked into buffer to create high and low positive samples (see x-axis; blank is just buffer). All possible combinations of capture antibody beads, detection antibody and analyte (CEA or GDF15 samples) were tested in a series of single-plex assays to verify lack of cross-reaction. (B.) To verify the p53 autoantibody assay could be fully multiplexed with the CEA and GDF15 sandwich immunoassays, a 3-plex VeraCode Bead™ assay was performed using a range of positive and negative sera/plasma for each analyte, and the results for each biomarker from the 3-plex assay (y-axis) were plotted by linear regression against the single-plex results (x-axis). To show the specificity of this metric, a linear regression between 3-plex measurements of GDF15 and p53 autoantibody, in which no correlation is expected, was also performed (lower right).