Enhancing a SARS-CoV-2 nucleocapsid antigen test sensitivity with cost efficient strategy through a cotton intermembrane insertion

Abstract

Lateral flow antigen tests have been widely used in the Covid-19 pandemic, allowing faster diagnostic test results and preventing further viral spread through isolation of infected individuals. Accomplishment of this screening must be performed with tests that show satisfactory sensitivity in order to successfully detect the target protein and avoid false negatives. The aim of this study was to create a lateral flow test that could detect SARS-CoV-2 nucleocapsid protein in low concentrations that were comparable to the limits of detection claimed by existing tests from the market. To do so, several adjustments were necessary during research and development of the prototypes until they were consistent with these criteria. The proposed alternatives of increasing the test line antibody concentration and addition of an intermembrane between the conjugate pad and the nitrocellulose membrane were able to increase the sensitivity four-fold and generate a new rapid test prototype called "lateral flow intermembrane immunoassay test" (LFIIT). This prototype showed an adequate limit of detection (2.0 ng mL^-1) while maintaining affordability and simplicity in manufacturing processes.

Figure 1

Hilab Flow, a device capable of performing lateral flow and vertical flow point of care tests.

Figure 2

P1 + 3.0 Limit of detection results for 3.75 ng mL⁻¹ of SARS-CoV-2 N protein. Replicates 1, 12 and 19: faint lines as reactive signals are indicated by the arrows. Replicate 8: no apparent test line (non-reactive). Control line shows the proper function of the test strips.

Figure 3

Traditional LFIA and a new proposed design called LFIIT. (A) Lateral view of a traditional lateral flow strip, showing the test components and assembly order. (B) Lateral view of a LFIIT prototype (insertion of an intermembrane), showing the test components and assembly order. Images not in scale and proportion.

Figure 4

LFIA prototypes signal comparison for a commercially available SARS-CoV-2 antigen control. (A) Representative image of the tests. P2: original P1 + 3.0 (3.0 mg mL⁻¹ antibody concentration on test line); P2 + 30 OD: original with 30 OD CGC; P2 + MF1: original with MF1 intermembrane; P2 + CF3: original with CF3 intermembrane; Control line shows the proper function of the test strips. (B) Bar plot showing statistical comparison between intensities of the test lines on different prototypes using a commercially available SARS-CoV-2 antigen control. (C) Signal comparison for 1:16 dilution of the same control material. Signal: Relative intensity units measured by Hilab Flow. # represents a significant difference compared to the P2 group. *Represents a significant difference compared to the P2 + MF1 group. Comparison between P2 + 30OD and P2 + CF3 showed no significant statistical difference. One-way ANOVA and Tukey post hoc test were carried out when appropriate for p < 0.05.

Figure 5

Prototype P2 + CF3 and P2 + 30 OD comparison in Limit of detection using 2.0 ng mL⁻¹ protein dilution. P2 + CF3: original prototype with a CF3 intermembrane addition. P2 + 30 OD: original with 30 OD CGC. Faint lines as reactive signals are indicated by the arrows. Control line shows the proper function of the test strips.

Figure 6

Final limit of detection results for LFIIT prototype with 2.0 ng mL⁻¹ of SARS-CoV-2 N protein. LFIIT lateral flow intermembrane immunoassay test (P2 + CF3). Faint lines as reactive signals are indicated by the arrows. Control line shows the proper function of the test strips.