A Simple Method To Prepare Oligonucleotide-Conjugated Antibodies For Multiplex Protein Detection

Abstracts: Fourth AACR International Conference on Frontiers in Basic Cancer Research; October 23-26, 2015; Philadelphia, PAWith rapid advances in nucleic acid detection technologies, genomics studies are being conducted in highly multiplexed formats. However, multiplex analysis is still a daunting task in the proteomics field; it would be of great interest to use genomics tools for protein detection. One way to accomplish this is to use oligonucleotide-conjugated antibodies to bind to specific protein targets, and to monitor conjugated-oligonucleotide levels rather than to measure protein levels directly. In this report we describe a simple method for preparing oligonucleotide-conjugated antibodies and discuss this method’s application in oligo extension reaction (OER) for multiplex protein detection. Conjugation is based on strain-promoted alkyne-azide cycloaddition (the Cu-free click reaction), in which the antibody is activated with a dibenzocyclooctyne (DBCO) moiety and linked covalently with an azide-modified oligonucleotide. The reaction condition and purification process were optimized to achieve maximum yield and best performance in the functional test. The OER assay employs a pair of antibody binders (two antibodies, each conjugated with its own oligonucleotide) developed for each protein target. The two oligonucleotides contain a six-base complementary region at their 3′ prime ends to allow annealing and extension by DNA synthesis enzymes to form a DNA template. The template is then detected by qPCR on the Biomark HD system. Distinct oligonucleotide sequences are assigned to different antibody binders to enable multiplex protein detection. When tested using recombinant proteins, some antibody binders, such as those specific to CSTB, MET, EpCAM and CASP3, had an estimated limit of detection (LOD) of about 0.06 pg/mL and a dynamic range of ≥5 logs. These antibody binders were used in multiplex protein detection in single cells using the C1™ system. The results in individual cells showed the expression of similar protein levels for CSTB and CASP3, but highly varied protein levels for EpCAM, CCNB1 and MUC1. This simple method for the preparation of oligonucleotide-conjugated antibodies is essential for the development of affordable multiplex protein assays, which has the potential to accelerate proteomics research.Citation Format: Haibiao Gong, Ilona Holcomb, Aik Ooi, Xiaohui Wang, Daniel Majonis, Marc Unger, Ramesh Ramakrishnan. A simple method to prepare oligonucleotide-conjugated antibodies for multiplex protein detection. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B18.