Rapid detection of antibodies in sera using multiplexed self-assembling bead arrays

Abstract

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 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.

HPV Serum Antibodies as Predictors of Survival and Disease Progression in Patients with HPV-Positive Squamous Cell Carcinoma of the Oropharynx

“…Bead array ELISAs were performed at Arizona State University essentially as described (11, 18, 19), with the following modifications (20). The in vitro translation products were captured on magnetic microspheres (BioRad, Hercules, CA), pooled, and blocked with HeteroBlock (Omega Biologicals, Bozeman, MT) diluted to 2 µg/mL into SeaBlock (Thermo Scientific, Rockford, IL).…”

Important Clues of Current Phosphoproteomic Approaches for Clinical Research

“…In addition to MS-based large-scale protein and peptide sequencing, other innovative approaches including self-assembling protein microarrays [78] and bead-based flow cytometry [79,80] to identify and quantify proteins and protein-protein interaction in a high throughput manner have furthered our understanding of the molecular mechanisms involved in diseases.…”

Antigen Discovery for Vaccines Using High‐Throughput Proteomic Screening Technologies

“…The nucleic acid programmable protein array (NAPPA) approach has been used to profile the antibody response to pathogens, including varicella zoster virus [45] and Pseudomonas aeruginosa [46], as well as the autoimmune antibody profile to discover markers for autoimmune disease [47] and cancer [48]. This self-assembling approach can also be used to prepare antigen-coated Luminex beads [49,50].…”