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. 2024 Apr 9;96(14):5407-5415.
doi: 10.1021/acs.analchem.3c05144. Epub 2024 Mar 13.

Overlaid Lateral Flow Immunoassay for the Simultaneous Detection of Two Variant-Specific SARS-CoV-2 Neutralizing Antibodies

Wanwisa Deenin  1   2 , Nanthika Khongchareonporn  2   3 , Kiat Ruxrungtham  4   5   6 , Chutitorn Ketloy  4   5   7 , Nattiya Hirankarn  8 , Kittikhun Wangkanont  9   10 , Sirirat Rengpipat  11 , Abdulhadee Yakoh  2   3 , Sudkate Chaiyo  2   3
Affiliations

Overlaid Lateral Flow Immunoassay for the Simultaneous Detection of Two Variant-Specific SARS-CoV-2 Neutralizing Antibodies

Wanwisa Deenin et al. Anal Chem. .

Abstract

COVID-19 vaccines have been provided to the general public to build immunity since the 2019 coronavirus pandemic. Once vaccinated, SARS-CoV-2 neutralizing antibodies (NAbs-COVID-19) are needed for excellent protection against COVID-19. However, monitoring NAbs-COVID-19 is complicated and requires hospital visits. Moreover, the resulting NAbs-COVID-19 are effective against different strains of COVID-19 depending on the type of vaccine received. Here, an overlaid lateral flow immunoassay (O-LFIA) was developed for the simultaneous detection of two NAbs-COVID-19 against different virus strains, Delta and Omicron. The O-LFIA was visualized with two T-lines with a single device using competition between the free antigen and the antigen-binding antibody. Angiotensin-converting enzyme 2 (ACE2) immobilized on the T-line binds to the antigen remaining after antibody binding. Under the optimum conditions, the proposed device exhibited 50% inhibition concentrations (IC50 values) of 45.1 and 53.6 ng/mL for the Delta and Omicron variants, respectively. Additionally, the proposed platform was applied to real-world samples of animal and human serum, and the developed immunoassay provided results that were in good agreement with those obtained with the standard method. In conclusion, this developed O-LFIA can be used as an alternative method to detect NAbs-COVID-19 and can be enabled for future advancements toward commercialization.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Schematic of the O-LFIA in each layer and photographs of the O-LFIAs with plastic cassettes for detecting the Delta and the Omicron NAbs-COVID-19 (A) and the immobilized biomaterial on the O-LFIA (B). LFIA positive (C) and negative (D) results when detecting Delta and Omicron NAbs-COVID-19.
Figure 2
Figure 2
Flow behavior of the O-LFIA using food coloring at different times (A). UV–Vis spectra of AuNPs and RBD/AuNPs (inset; TEM image of AuNPs) (B). Response after simultaneous detection of two variants of NAbs-COVID-19 using the O-LFIA at concentrations of 0–200 ng/mL (C). Color intensity comparison at concentrations of 0 (left), 50 (middle), and 200 (right) ng/mL for the detection of two variants of NAbs-COVID-19 (D) (n = 3).
Figure 3
Figure 3
Photographs of the O-LFIA responses with different dilutions of Delta and Omicron NAbs-COVID-19 (A). The relationship between % inhibition and different dilutions of ΔNAbs (B) and oNAbs (C). The linear relationship between % inhibition and ΔNAb (inset B) and oNAb (inset C) is also shown (n = 3).
Figure 4
Figure 4
Evaluation of vaccine effectiveness in monkey serum at days 0 (D0), 30 (D30), and 60 (D60) using the O-LFIA and microneutralization assay (MN50 titer).
Figure 5
Figure 5
Schematic diagram of the NAb-COVID-19 O-LFIA procedure (A), vaccine effectiveness determined with human serum using an ELISA test kit (B) and the O-LFIA (C), and correlation analysis for estimating the % inhibition of NAbs-COVID-19 at different NAb-COVID-19 levels using an ELISA test kit and the O-LFIA device (D).
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