Quantification of siRNA-Antibody Conjugates in Biological Matrices by Triplex-Forming Oligonucleotide ELISA

Nucleic Acid Therapeutics
Sara C HumphreysBrooke M Rock

Abstract

The potential repertoire of short interfering RNA (siRNA) therapeutics is expanding as targeting strategies evolve. One approach to enable organ-specific delivery has been to directly conjugate siRNA to a monoclonal antibody (siRNA-mAb), analogous to antibody-drug conjugates. Detection of intact siRNA-mAb conjugates presents a bioanalytical challenge given that certain synthetic nucleotide chemical modifications and low-temperature requirements render common oligonucleotide detection assays, such as reverse transcription-polymerase chain reaction, incompatible with the immunoassay component. To circumvent these issues, we developed a triplex-forming oligonucleotide ELISA using locked nucleic acid (LNA) containing oligonucleotide probes. We demonstrate that the incorporation of these LNAs allow for an enrichment and immobilization of siRNA directly conjugated to an antibody at nondenaturing temperatures. Without further requirement for extraction or amplification, we can sensitively and specifically detect intact siRNA-mAb conjugates in complex matrices such as serum and tissue homogenate.

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Citations

Jan 16, 2020·Antibodies·Donmienne LeungYiqing Feng
Aug 11, 2019·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Brooke M Rock, Robert S Foti
Apr 23, 2021·Journal of the American Society for Mass Spectrometry·Iain D G Campuzano, Wendy Sandoval

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Methods Mentioned

BETA
size-exclusion
electrophoresis
nuclear magnetic resonance
PCR
ELISA

Software Mentioned

MassLynx
PMI Intact

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