Antibody Microarrays Utilizing Site-Specific Antibody–Oligonucleotide Conjugates

Abstract

Protein arrays are typically made by random absorption of proteins to the array surface, potentially limiting the amount of properly oriented and functional molecules. We report the development of a DNA encoded antibody microarray utilizing site-specific antibody–oligonucleotide conjugates that can be used for cell immobilization as well as the detection of genes and proteins. This technology allows for the facile generation of antibody microarrays while circumventing many of the drawbacks of conventionally produced antibody arrays. We demonstrate that this method can be used to capture and detect SK-BR-3 cells (Her2+ breast cancer cells) at concentrations as low as 102 cells/mL (which is equivalent to 10 cells per 100 µL array) without the use of microfluidics, which is 100- to 105-fold more sensitive than comparable techniques. Additionally, the method was shown to be able to detect cells in a complex mixture, effectively immobilizing and specifically detecting Her2+ cells at a concentration of 102 SK-BR-3 cells/mL in 4 × 106 white blood cells/mL. Patients with a variety of cancers can have circulating tumor cell counts of between 1 and 103 cells/mL in whole blood, well within the range of this technology.

Rapid fluorescence immunoassay of benzo[a]pyrene in mainstream cigarette smoke based on a dual-functional antibody–DNA conjugate

“…To simplify the immunoassay procedure, in this study, we developed a RFIA method using synthetic covalent BaP antibody-DNA conjugates for recognition and signal amplication by using the reported detection strategy of antibody-DNA complexes. 25,27,28 As illustrated in Fig. 1, the -NH 2 -modied reporter DNA and BaP antibody were activated with sulfo-SMCC and SATA, respectively; and the two activated molecules then react to form an immune complex covalently coupled with reporter DNA and BaP antibodies for immunodetection of BaP.…”

Site-selective orientated immobilization of antibodies and conjugates for immunodiagnostics development

“…The oligonucleotide labelling strategies are usually non-selective. [193,194] However, the site-specificity during immobilization can be achieved via unnatural amino acid [195,196] or oligonucleotide labelled protein G [197]. The immobilization via oligonucleotide hybridization is simple but efficient approach and suitable for surface regeneration in a timely manner.…”

“…In addition, antibody with site-specifically labelled oligonucleotides can be produced using p -acetylphenylalanine incorporated antibody and aminoxy-functionalized single-stranded oligonucleotide, which resulted in a stable oxime linkage. [195,196] It has been shown that few such amino acids, such as p -benzoyl- L -phenylalanine, can work as photo-reactive crosslinker for covalently coupling antibody and its affinity partner, where the photo-reactive crosslinker could be incorporated into either antibody or its affinity partner. [169,202]…”

Nucleic Acid Bioconjugates in Cancer Detection and Therapy

“…The HER2‐positive cells were shown to bind specifically to the antibody–DNA microarray with a >1000‐fold difference in signal relative to the HER2‐negative cells (MDA‐MB‐231) . This type of microarray may be useful in screening for biological markers of selected tumor types that may result in the early detection and treatment of cancer.…”