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. 2023 Apr;415(10):1897-1904.
doi: 10.1007/s00216-023-04600-2. Epub 2023 Feb 23.

Quantitative performance of digital ELISA for the highly sensitive quantification of viral proteins and influenza virus

Takema Hasegawa  1 , Sachie Shibayama  2 , Yukiko Osumi  2 , Hiroshi Sentsui  3 , Megumi Kato  2
Affiliations

Quantitative performance of digital ELISA for the highly sensitive quantification of viral proteins and influenza virus

Takema Hasegawa et al. Anal Bioanal Chem. 2023 Apr.

Abstract

A single-molecule assay (SiMoA) using a digital enzyme-linked immunosorbent assay (ELISA) has been attracting attention as a promising method that can detect viruses with ultra-high sensitivity. However, the quantitative application of digital ELISA has not been adequately reported. Therefore, in this study, we first evaluated the linearity and sensitivity of digital ELISA using a Certified Reference Material of C-reactive protein (NMIJ CRM 6201-c) as a quality control material. Next, we originally screened those antibody pair that are suitable for detecting recombinant viral proteins of influenza A virus, nucleoprotein (NP), and hemagglutinin (HA), and established the measurement system. Under optimized conditions, the limit of detection (LOD) of NP and HA was 0.59 fM and 0.99 fM, and the coefficient of determination, R2, was 0.9998 and 0.9979, respectively. Two subtypes of influenza virus, A/Puerto Rico/8/1934 (H1N1) [PR8] and A/Panama/2007/99 (H3N2) [Pan99], were also quantified under established conditions, and the LOD of PR8 was 3.1 × 102 PFU/mL on targeting NP and 7.4 × 102 PFU/mL on targeting HA. The LOD of Pan99 was 5.3 × 102 PFU/mL on targeting NP. The specificity and robustness of the recombinant viral protein and influenza virus measurements using digital ELISA were also evaluated. Our measurement system showed enough specificity to discriminate the viral subtypes properly and showed sufficient inter- and intra-assay variations for both measurements of recombinant viral proteins and viruses, except for NP-targeting virus measurement.

Keywords: Digital ELISA; Influenza virus; Quantitative efficacy; SiMoA.

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References

    1. Saunders-Hastings PR, Krewski D. Reviewing the history of pandemic influenza: understanding patterns of emergence and transmission. Pathogens. 2016;5(4):66. https://doi.org/10.3390/pathogens5040066 . - DOI - PubMed - PMC
    1. Hutchinson EC. Influenza virus. Trends Microbiol. 2018;26(9):809–10. https://doi.org/10.1016/j.tim.2018.05.013 . - DOI - PubMed
    1. Harris A, Cardone G, Winkler DC, Heymann JB, Brecher M, White JM, et al. Influenza virus pleiomorphy characterized by cryoelectron tomography. Proc Natl Acad Sci. 2006;103(50):19123–7. https://doi.org/10.1073/pnas.0607614103 . - DOI - PubMed - PMC
    1. Aoki FY, Macleod MD, Paggiaro P, Carewicz O, El Sawy A, Wat C, et al. Early administration of oral oseltamivir increases the benefits of influenza treatment. J Antimicrob Chemother. 2003;51(1):123–9. https://doi.org/10.1093/jac/dkg007 . - DOI - PubMed
    1. Russo A, Minichini C, Starace M, Astorri R, Calò F, Coppola N, et al. Current status of laboratory diagnosis for COVID-19: a narrative review. Infect Drug Resist. 2020;13:2657–65. https://doi.org/10.2147/IDR.S264020 . (Published 2020 Aug 3). - DOI - PubMed - PMC

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