Antibody detection assays, such as ELISA, immunoprecipitation, and radioimmunoassays are important tools to diagnose diseases such as Type 1 Diabetes (T1D)1–3. When performed manually, these common yet labor-intensive assays can take up to 22 hours and are subject to inconsistency between technicians and laboratories. In addition, gold standard radioimmunoassays require radioactive reagents, which increase assay costs and risk of personnel exposure to hazardous reagents, and offer minimal multiplexing capability. As a result, they are not feasible for large population antibody screening.
Antibody Detection by Agglutination-PCR (ADAP) differs from traditional immunoassays in that it is a pure solution-phase assay and well-suited to detecting sensitive antibodies. The assay utilizes a pair of antigen-DNA conjugates to probe the sample target antibodies. If present, agglutination between the antibody and antigen, and ligation, leads to formation of a specific DNA amplicon detected by qPCR.
In this application note, we describe multiplexed ADAP detection of Insulin (Ins), Islet Antigen 2 (IA-2), and Glutamic Acid Decarboxylase (GAD) using the ADAP T1D reagent chemistry kit and readily available quantitative (qPCR) instruments. The ADAP T1D reagent chemistry kit consists of four reagents, each labeled for agglutination, ligation, pre-amplification, and qPCR primer quantification, along with positive and negative controls. Up to four antibodies may be detected per sample in a 384-well quantification plate. Using the ADAP T1D reagent chemistry kit, the autoantibodies can be detected in the blood and serum of pre-symptomatic individuals with T1D, thus offering an avenue for early disease management, such as timely glycemic control, to prevent metabolic complications and reduce the onset of potentially fatal diabetes ketoacidosis.
The workflow is automated using the ADAP STAR assay ready workstation from Hamilton Company (see workflow and deck layout at end). The ADAP STAR is based on the Company’s Microlab® STAR™ liquid handling workstation, and is configured with CO-RE® gripper paddles and the iSWAP robotic transport arm for hands-free plate and lid movements. Microlab VENUS® software in ADAP STAR was optimized and pre-programmed for all steps. Using this automated workflow, up to 80 samples, along with necessary controls, may be analyzed in one automated run. We demonstrate that the ADAP STAR enables increased throughput and facilitates sample-to-answer within three hours.