Ultrasensitive protein-level detection for respiratory infectious viruses

Abstract

Influenza virus, adenovirus, and respiratory syncytial virus cause major respiratory infections. These infections have similar initial symptoms making it difficult to differentiate them based on symptoms alone. PCR is currently used as the standard diagnostic test for these infections, however, it has its limitations such as non-specific and false-negative amplifications, high cost, and the inability to distinguish between a live or dead virus. Therefore, there is a need for alternative diagnostic methods that focus on protein. Here, we introduce TN-cyclon™, which is an enzyme-linked immunosorbent assay combined with thio-nicotinamide adenine dinucleotide cycling to amplify signals, rather than the protein itself. Using this method, we were able to detect extremely low levels of viruses such as influenza A, influenza B, adenovirus, and RS virus, with LODs of 2.96 × 10^-18 moles/assay, 2.98 × 10^-18 moles/assay, 2.36 × 10^-18 moles/assay, and 3.55 × 10^-18 moles/assay, respectively. Furthermore, we successfully detected viruses diluted with extract buffer, with a significant difference to the blank at concentrations of 3 pfu/mL for influenza A, 1000 pfu/mL for influenza B, 43.8 pfu/mL for adenovirus, and 125 pfu/mL for RS virus. This shows that our low-cost and easy-to-use technique has sufficient sensitivity in diagnosing respiratory infections.

Keywords: adenovirus; influenza virus; protein detection assay; respiratory syncytial virus; thio-NAD cycling; ultrasensitive ELISA.

Figure 1

Schematic diagram of thio-NAD cycling ELISA. The signal generated by alkaline phosphatase is amplified through redox cycling via the enzyme 3α-hydroxysteroid dehydrogenase(3α-HSD) and its coenzymes, thio-NAD and NADH.

Figure 2

A thio-NAD cycling ELISA was used to measure the calibration curves for the nucleoproteins of influenza A (A) and B (B), the hexon protein of adenovirus (C), and the fusion protein of RS virus (D). The absorbance was measured at the following time points of thio-AND cycling: 55 min for influenza A, 55 min for influenza B, 60 min for adenovirus, and 50 min for RS virus (n = 3 each). Each of the time points was determined before the saturation of the reaction absorbance. The antigen was applied within a range of 31.25 – 1000 pg/mL.

Figure 3

Detection of nucleoproteins in surfactant-inactivated influenza virus type A. Three datasets were measured by three investigators and are presented as (A–C). Triplicate measurements were performed at 60 min of thio-NAD cycling by each investigator. All datasets show that the signals were significantly higher than the blank values at concentrations over 3.13 pfu/mL (*P < 0.05, **P < 0.01).

Figure 4

Detection of nucleoproteins in surfactant-inactivated influenza virus type B. Three datasets were measured by three investigators and are presented as (A–C). Triplicate measurements were performed at 60 min of thio-NAD cycling by each investigator. All datasets show that the signals were significantly higher than the blank values at concentrations over 1000 pfu/mL (*P < 0.05, **P < 0.01).

Figure 5

Detection of hexon proteins in surfactant-inactivated adenovirus. Three datasets were measured by three investigators and are presented as (A–C). Triplicate measurements were performed at 60 min of thio-NAD cycling by each investigator. All datasets show that the signals were significantly higher than the blank values at the concentrations over 43.8 pfu/mL (*P < 0.05, **P < 0.01).

Figure 6

Detection of fusion proteins in surfactant-inactivated RS virus. Three datasets were measured by three investigators and are presented as (A–C). Triplicate measurements were performed at 60 min of thio-NAD cycling by each investigator. All datasets show that the signals were significantly higher than the blank values at concentrations over 125 pfu/mL (*P < 0.05, **P < 0.01).