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

• Published in: Molecular therapy. Nucleic acids 2023-06, Vol.32, p.445-453
• Main Authors: Luo, Dengwang, Wu, Zhanfeng, Wang, Daming, Zhang, Jieli, Shao, Fei, Wang, Shuo, Cestellos-Blanco, Stefano, Xu, Dawei, Cao, Yuhong
• Format: Article
• Language: English
• Subjects: dot-blot assay; double-stranded RNA; immune response; In vitro-transcribed mRNA; lateral flow strip assay; MT: Oligonucleotides: Diagnostics and Biosensors; Original; rapid and sensitive detection
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2023.04.005

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. [Display omitted] Cao and colleagues develop a rapid and sensitive lateral flow strip assay (LFSA) for the detection of dsRNA contaminants in in vitro-transcribed (IVT) mRNA. This straightforward method is 16 times faster than the gold-standard dot-blot assay, while also constituting an early warning tool for the prevention of potential immunogenicity from dsRNA.