Ultrasensitive Antibody Detection by Agglutination-PCR(ADAP)

摘要

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 spanning5–6 orders of magnitude. Using ADAP, we detected anti-thyroglobulinautoantibodies from human patient plasma with a 1000-fold increasedsensitivity over an FDA-approved radioimmunoassay. Finally, we demonstratethe multiplexability of ADAP by simultaneously detecting multipleantibodies in one experiment. ADAP’s combination of simplicity,sensitivity, broad dynamic range, multiplexability, and use of standardPCR protocols creates new opportunities for the discovery and detectionof antibody biomarkers.