Rapid detection of antibody immunity in serum or plasma, whether to pathogenic antigens,
tumor antigens, or autoimmune antigens, is critical for diagnosis, monitoring, and
biomarker assessment of the immune response. Individual or multiplexed ELISAs that
use purified recombinant proteins are dependent on a priori protein purification,
a labor-intensive process that may take months to obtain proteins of sufficient purity
and stability for serologic assays. We developed a programmable multiplexed immunoassay
for the rapid monitoring of humoral immunity using the Luminex suspension bead array
platform. In this approach, epitope-tagged antigens (GST- or FLAG-tagged) are expressed
using in vitro transcription and translation, and captured onto anti-epitope-coupled
Luminex SeroMap beads. The antigen-loaded beads are mixed, serum is added, and human
IgG is detected with standard secondary detection reagents. By coupling high-throughput
DNA preparation of cDNA ORFs with antigen expression/capture, we demonstrate that
71/72 (98.6%) of GST-tagged proteins can be expressed and specifically detected on
the bead ELISA. Detection of antibodies to the test viral antigen EBNA-1 in human
sera is highly reproducible, with intra-assay variation of 3-8%, inter-assay variation
of 5%, and with stability over 11 months. The specificity and limits of detection
of the bead ELISAs for the tumor antigen p53 are comparable to both standard protein
ELISAs and plate-based programmable (RAPID) ELISAs, and are also comparable to the
detection of directly-conjugated p53 protein. Multiplexing a panel of analytes does
not impair the sensitivity of antibody detection. Immunity to a panel of EBV-derived
antigens (EBNA-1, EBNA-3A, EBNA-3B, and LMP-2) is specifically and differentially
detected within healthy donor sera. This method allows for rapid conversion of ORFeome-derived
cDNAs to a multiplexed bead ELISA to detect antibody immunity to both infectious and
tumor antigens.