Dual Recognition Element Lateral Flow Assay Toward Multiplex Strain Specific Influenza Virus Detection

Abstract

Different influenza virus strains have caused a number of recent outbreaks killing scores of people and causing significant losses in animal farming. Simple, rapid, sensitive, and specific detection of particular strains, such as a pandemic strain versus a previous seasonal influenza, plays a crucial role in the monitoring, controlling, and management of outbreaks. In this paper we describe a dual recognition element lateral flow assay (DRELFA) which pairs a nucleic acid aptamer with an antibody for use as a point-of-care platform which can detect particular strains of interest. The combination is used to overcome the individual limitations of antibodies' cross-reactivity and aptamers' slow binding kinetics. In the detection of influenza viruses, we show that DRELFA can discriminate a particular virus strain against others of the same subtype or common respiratory diseases while still exhibiting fast binding kinetic of the antibody-based lateral flow assay (LFA). The improvement in specificity that DRELFA exhibits is an advantage over the currently available antibody-based LFA systems for influenza viruses, which offer discrimination between influenza virus types and subtypes. Using quantitative real-time PCR (qRT-PCR), it showed that the DRELFA is very effective in localizing the analyte to the test line (consistently over 90%) and this is crucial for the sensitivity of the device. In addition, color intensities of the test lines showed a good correlation between the DRELFA and the qRT-PCR over a 50-fold concentration range. Finally, lateral flow strips with a streptavidin capture test line and an anti-antibody control line are universally applicable to specific detection of a wide range of different analytes.

Figure 1

Schematic diagram of a DRELFA for detection of a virus. An anti-virus antibody (Y-shape) is conjugated with GNPs (golden oval). An aptamer (black line) has a biotin (blue star) at its 5′-or 3′-end. (a) In the presence of the specific virus, both the aptamer and the gold nanoparticle (GNP)-conjugated antibody bind to the virus (brown-textured sphere) and the biotin on the aptamer enables the complex to be bound onto the streptavidin test line (purple line) and the detection can be made by the color of GNPs. (b) Absence of the specific virus would result in the capture of GNPs on the secondary antibody control line (red line) only.

Figure 2

(a) Binding of an RNA aptamer to whole virus particles of 3 different strains of the influenza A H3N2 using ELONA: A/Panama/2007/99 (Panama/99), A/Aichi/2/68 (Aichi/68), and A/Udorn/307/72 (Udorn/72). The aptamer can discriminate one strain from others of the H3N2 subtype. (b) A monoclonal antibody shows binding to all three strains of the influenza A H3N2: Panama/99, Aichi/68, and Udorn/72. In all cases 7 × 10⁸ virus particles were added to each well for immobilization of the virus on the microtiter plate.

Figure 3

Specificity of LFA’s (a) DRELFA: The sample with Panama/99 has 2 lines showing visual detection of this virus strain while measurements for Udorn/72 and Aichi/68 samples have only 1 line (control quality) indicating that this DRELFA for Panama/99 virus does not have any cross reactivity with other strains of this H3N2 subtype. (b) Antibody-LFA: all 3 samples of the influenza A H3N2 displayed 2 lines showing visual detection of the viruses, indicating that the conventional LFA does not differentiate between the three virus strains of the H3N2 subtype. The measurements were performed with samples of 10⁸ virus particles. The sample volume was 100 μL.

Figure 4

(a) Distribution of the virus particles on the lateral flow membranes were analyzed using qRT-PCR. It shows that consistently >90% of the virus particles are captured on the test line. (b) Comparison of the intensity of the test lines’ signals to the virus copies obtained from the RNA using qRT-PCR at different virus dilutions. The image intensity and the RNA copy number have a correlation coefficient of 0.995.