Titration of trace amounts of immunoglobulins in a microarray-based assay with magnetic labels

The existing immunoassay format that combines the electrophoretic collection of charged analytes on an antibody microarray with the detection of the bound analytes by magnetic beads coated with secondary antibodies displays extreme sensitivity and speed, but suffers from low precision because of high signal scatter and low signal-to-concentration ratio. Here we report three innovations that substantially improve the precision of this method and enable quantitative measurements of analyte concentrations as low as 10 fg/ml. The improvements were achieved by (i) employing parallel titration of analytes by measuring signal response to a series of sample dilutions with increasing analyte concentration, (ii) internally normalizing the signal (by relating signal intensity to that of positive controls on the same microarray) and (iii) taking measurements in the linear range of the calibration curve at concentrations close to the limit of detection. This improved method was used to quantitatively measure in human serum the titer of immunoglobulins specific to antigens secreted by Mycobacterium tuberculosis.