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.

中文翻译：

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通过三链体形成寡核苷酸ELISA定量分析生物基质中的siRNA抗体。

随着靶向策略的发展，短干扰RNA（siRNA）治疗剂的潜在功能正在扩展。一种实现器官特异性递送的方法是直接将siRNA与单克隆抗体（siRNA-mAb）偶联，类似于抗体-药物偶联物。鉴于某些合成核苷酸的化学修饰和低温要求使得常见的寡核苷酸检测测定（例如逆转录-聚合酶链反应）与免疫测定成分不兼容，完整的siRNA-mAb缀合物的检测提出了生物分析挑战。为了解决这些问题，我们使用含有寡核苷酸探针的锁定核酸（LNA）开发了形成三链体的寡核苷酸ELISA。我们证明这些LNA的纳入允许在非变性温度下直接与抗体偶联的siRNA的富集和固定化。无需进一步提取或扩增，我们就可以灵敏和特异性地检测复杂基质（例如血清和组织匀浆）中完整的siRNA-mAb偶联物。