Oligonucleotide-mediated quantitative multiplexed immunoassays

Methods and compositions for quantitative immunoassays are provided, in which ligand-conjugated probes are used to label samples and ligand-surfaced microspheres are used as quantitative reference standards. Certain embodiments provide a method of quantitative flow cytometry where ligands are oligonucleotides, and a sample comprising one or more cells is contacted with a hybridized antibody::fluorophore labeled targeting construct to label the cells, and the labeled cells are analyzed. In some embodiments, a population of quantitative labeled oligospheres labeled with the same fluorescent label as the cells is analyzed using the flow cytometer and used to create a quantitative standard curve of cytometer intensity versus molecules fluorescent label per oligosphere event. A standard curve trendline is established and used to determine the molecules of fluorescent label per cellular event for the antigen-positive cell populations. Based on molecules of fluorescent label per cell, the amount of Antibody Binding per Cell (ABC) is quantified.