Dataset Information

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 ?L 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.

SUBMITTER: Tsai CT

PROVIDER: S-EPMC4819452 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP).

Tsai Cheng-Ting CT Robinson Peter V PV Spencer Carole A CA Bertozzi Carolyn R CR

ACS central science 20160216 3

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 ...[more]

PMID: 27064772

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Project description:BackgroundThe accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR.ResultsA liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose-response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was <6?% for repeatability and <2?% for reproducibility. The assay detection limit was 13 fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed.ConclusionsThe ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the liposomes allows nonspecific DNA in the assay medium to be degraded with DNase I prior to quantification of the encapsulated reporter by PCR, which reduces false-positive results and improves quantitative accuracy. The ability to encapsulate multiple reporters per liposome also helps overcome the effect of polymerase inhibitors present in biological specimens. Finally, the biotin-labeled liposome detection reagent can be coupled through a NeutrAvidin bridge to a multitude of biotin-labeled probes, making ILPCR a highly generic assay system.

Project description:In oncology, liquid biopsy is used in the detection of next-generation analytes, such as tumor cells, cell-free nucleic acids and exosomes in peripheral blood and other body fluids from cancer patients. It is considered one of the most advanced non-invasive diagnostic systems to enable clinically relevant actions and implement precision medicine. Medical actions include, but are not limited to, early diagnosis, staging, prognosis, anticipation (lead time) and the prediction of therapy responses, as well as follow-up. Historically, the applications of liquid biopsy in cancer have focused on circulating tumor cells (CTCs). More recently, this analysis has been extended to circulating free DNA (cfDNA) and microRNAs (miRNAs or miRs) associated with cancer, with potential applications for development into multi-marker diagnostic, prognostic and therapeutic signatures. Liquid biopsies avoid some key limitations of conventional tumor tissue biopsies, including invasive tumor sampling, under-representation of tumor heterogeneity and poor description of clonal evolution during metastatic dissemination, strongly reducing the need for multiple sampling. On the other hand, this approach suffers from important drawbacks, i.e., the fragmentation of cfDNA, the instability of RNA, the low concentrations of certain analytes in body fluids and the confounding presence of normal, as well as aberrant DNAs and RNAs. For these reasons, the analysis of cfDNA has been mostly focused on mutations arising in, and pathognomonicity of, tumor DNA, while the analysis of cfRNA has been mostly focused on miRNA patterns strongly associated with neoplastic transformation/progression. This review lists some major applicative areas, briefly addresses how technology is bypassing liquid biopsy limitations, and places a particular emphasis on novel, PCR-free platforms. The ongoing collaborative efforts of major international consortia are reviewed. In addition to basic and applied research, we will consider technological transfer, including patents, patent applications and available information on clinical trials aimed at verifying the potential of liquid biopsy in cancer.

Dataset Information

Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP).

Publications

Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP).

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