Described are methods for simultaneous detection and quantifying
multiple target analytes, including immunoglobulin isotypes and sub-classes, single and multiple
protein antibodies within a
test sample contained in a single reaction vessel. Such methods use reaction wells as on a multi-well plate, each single well comprising microarrays of calibration spots, each having a predetermined quantity of a target
analyte; and capture spots, each having multiple agent antibodies, including isotypes and subclasses that specifically bind the target analytes. The captured analytes and the calibration spots are detected with fluorescently labeled antibodies specific for each different target
analyte. Calibration spots generate calibration curves for quantitative determinations of different target analytes. Also described are methods for detecting and quantifying biomarkers, therapeutic proteins and patient derived antibodies; the use of secondary reagents to determine immunoglobulin classes Ig G, A, M, E and sub-classes including IgG1, IgG2, IgG3, IgG4 and IgA. The intensity of each fluorescent
signal allows measurement of a specific immune response to a
therapeutic protein and associated analytes; interrogates neutralizing effects of patient antibodies on therapeutic proteins, e.g.,
insulin therapy.