Lateral flow immunoassay for rapid and sensitive detection of dsRNA contaminants in in vitro-transcribed mRNA products

Dengwang Luo; Zhanfeng Wu; Daming Wang; Jieli Zhang; Fei Shao; Shuo Wang; Stefano Cestellos-Blanco; Dawei Xu; Yuhong Cao

doi:10.1016/j.omtn.2023.04.005

Lateral flow immunoassay for rapid and sensitive detection of dsRNA contaminants in in vitro-transcribed mRNA products

Mol Ther Nucleic Acids. 2023 Apr 8:32:445-453. doi: 10.1016/j.omtn.2023.04.005. eCollection 2023 Jun 13.

Authors

Dengwang Luo^{1

2}, Zhanfeng Wu^{3

4}, Daming Wang^{5

6

7}, Jieli Zhang⁷, Fei Shao^{4

8}, Shuo Wang⁸, Stefano Cestellos-Blanco⁹, Dawei Xu², Yuhong Cao^{2

10}

Affiliations

¹ Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
² CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Chinese Academy of Sciences, Beijing 100190, China.
³ Beijing National Laboratory for Molecular Sciences, Key Laboratory of Molecular Nanostructure and Nanotechnology, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
⁴ University of Chinese Academy of Sciences, Beijing 100049, China.
⁵ Academy for Engineering and Technology, Fudan University, Shanghai 200433, China.
⁶ Suzhou Institute of Biomedical Engineering and Technology (SIBET), Chinese Academy of Sciences, Suzhou, Jiangsu 215163, China.
⁷ Anbio Biotechnology Company, Xiamen, Fujian 361026, China.
⁸ CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
⁹ Department of Materials Science and Engineering, University of California, Berkeley, Berkeley, CA 94720, USA.
¹⁰ School of Nanoscience and Technology, University of Chinese Academy of Sciences, Beijing 100049, China.

Abstract

High purity is essential in mRNA-based therapeutic applications. A major contaminant of in vitro-transcribed (IVT) mRNA manufacturing is double-stranded RNA (dsRNA), which can induce severe anti-viral immune responses. Detection methods, such as agarose gel electrophoresis, ELISA, and dot-blot assay, are used to detect the existence of dsRNA in IVT mRNA products. However, these methods are either not sensitive enough or time-consuming. To overcome these challenges, we develop a rapid, sensitive, and easy-to-implement colloidal gold nanoparticle-based lateral flow strip assay (LFSA) with sandwich format for the detection of dsRNA from IVT process. dsRNA contaminant can be determined visually on the test strip or quantitatively with a portable optical detector. This method allows for a 15 min detection of N1-methyl-pseudouridine (m1Ψ)-containing dsRNA with a detection limit of 69.32 ng/mL. Furthermore, we establish the correlation between the LFSA test results and the immune response caused by dsRNA in mice. The LFSA platform allows the rapid, sensitive, and quantitative monitoring of purity in massive IVT mRNA products and aids for the prevention of immunogenicity by dsRNA impurities.

Keywords: In vitro-transcribed mRNA; MT: Oligonucleotides: Diagnostics and Biosensors; dot-blot assay; double-stranded RNA; immune response; lateral flow strip assay; rapid and sensitive detection.