Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP)

Abstract

Antibodies are widely used biomarkers for the diagnosis of many diseases. Assays based on solid-phase immobilization of antigens comprise the majority of clinical platforms for antibody detection, but can be undermined by antigen denaturation and epitope masking. These technological hurdles are especially troublesome in detecting antibodies that bind nonlinear or conformational epitopes, such as anti-insulin antibodies in type 1 diabetes patients and anti-thyroglobulin antibodies associated with thyroid cancers. Radioimmunoassay remains the gold standard for these challenging antibody biomarkers, but the limited multiplexability and reliance on hazardous radioactive reagents have prevented their use outside specialized testing facilities. Here we present an ultrasensitive solution-phase method for detecting antibodies, termed antibody detection by agglutination-PCR (ADAP). Antibodies bind to and agglutinate synthetic antigen–DNA conjugates, enabling ligation of the DNA strands and subsequent quantification by qPCR. ADAP detects zepto- to attomoles of antibodies in 2 μL of sample with a dynamic range spanning 5–6 orders of magnitude. Using ADAP, we detected anti-thyroglobulin autoantibodies from human patient plasma with a 1000-fold increased sensitivity over an FDA-approved radioimmunoassay. Finally, we demonstrate the multiplexability of ADAP by simultaneously detecting multiple antibodies in one experiment. ADAP’s combination of simplicity, sensitivity, broad dynamic range, multiplexability, and use of standard PCR protocols creates new opportunities for the discovery and detection of antibody biomarkers.

Dual-reporter SERS-based biomolecular assay with reduced false-positive signals

“…ELISA's reliability results from the use of two affinity reagents to capture and sandwich the target protein as a requisite for generating a reporter signal, an approach that reduces false-positive signals. This "sandwich" assay structure has inspired many groups to develop hybrid versions of ELISA that incorporate novel reporting techniques such as PCR, optical detection, and surface-enhanced Raman spectroscopy (SERS) to further improve sensitivity (3)(4)(5)(6)(7).…”

Antibody detection by agglutination–PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis

“…The induced proximity enables ligation of DNA fragments to form a full-length DNA amplicon, which can then be quantified by qPCR. As reported previously, this amplification permits detection of antigen-specific antibodies at high zeptomole levels in 1-μL samples (21). Since ligation is only triggered following a productive antibody-antigen interaction, ADAP does not require washing steps to remove unbound antigen-DNA conjugates and is thus well-equipped to detect low-affinity antibodies.…”

“…Here we report an ultrasensitive OF HIV antibody detection method based on Antibody Detection by Agglutination-PCR (ADAP) technology ( Fig. 1) (21). The ADAP platform, similar in nature to proximity ligation assay (PLA) (22), leverages multivalent binding of antibodies to drive the agglutination of antigen-DNA conjugates.…”

Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies

“…Ligation is terminated by the addition of a uracil excision mixture, which selectively degrades the bridge oligonucleotide. The ligation mixture is subsequently preamplified across the newly formed DNA junction to increase signal-to-background ratio and reproducibility (12,35) before quantification by qPCR. In c-PLA, ligation products are not formed by a direct junction of proximity probes but rather, by the formation of a circle when two free connector oligonucleotides are joined in two distinct ligation events facilitated by target-bound proximity probes.…”