Boosting lateral flow immunoassay sensitivity by increasing antigen-antibody interaction time and test line antibody concentration

Lateral flow immunoassays (LFIAs) are useful tools for point of care diagnostic testing. Improving their sensitivity can help prevent the spread of viruses like SARS-CoV-2 by detecting infections earlier. In effort of lowering the limit of detection of their COVID-19 rapid antigen test, scientists at Hilab have developed a rapid test prototype called “lateral flow intermembrane immunoassay test” that brings the limit of detection to 2.0 ng/mL of SARS-CoV-2 nucleocapsid protein¹.

How do lateral flow immunoassays work?

Lateral flow assays have enabled the rapid detection of SARS-CoV-2. Compared to other diagnostic tests, rapid tests are low cost, fast, and can be done right at home. When a sample is loaded onto the test strip, it flows across the test strip where it has the chance to interact with various antibodies to produce a readout of the results. The test strip is composed of the following components:

Sample pad: where the sample is loaded
Conjugate pad: contains nanoparticle-antibody conjugates that bind any antigen present in the sample
Nitrocellulose membrane: contains the test line (which binds the nanoparticle-antigen complex) and control line (which binds the nanoparticle, with or without antigen present, to ensure test validity)
Wicking pad: ensures even flow of sample across the test strip

In a study published in Nature Scientific Reports, the diagnostics and digital health company, Hilab, optimized their existing COVID-19 test to reduce its limit of detection1. The original test had a limit of detection at 7.5 ng/mL of nucleocapsid protein and relied on a two-step verification process for the results including visual inspection and AI detection.

The tests described below were built using colloidal gold nanoparticles, COVID-19 nucleoprotein monoclonal antibody (test line), and goat anti-mouse IgG antibody colloidal gold (control line) from Arista Biologicals, a Fortis Life Sciences® company. After running the tests, the authors used visual and instrument detection to assess the test results.

Strategy 1: Increasing lateral flow assay sensitivity by increasing test line antibody concentration

To increase the sensitivity of their lateral flow assay, the authors first created a prototype that doubled the amount of the test line antibody to 3.0 mg/mL. Using this prototype, they screened four concentrations of nucleocapsid protein (100 ng/mL, 10 ng/mL, 5 ng/mL, and 1 ng/mL). Based on the positive and negative results at different concentrations, they found that the limit of detection is likely between 5.0 ng/mL and 1 ng/mL.

Since they doubled the test line antibody concentration, the authors suspected that the limit of detection would be half of the initial limit of detection of 7.5 ng/mL. As defined by the FDA, the limit of detection is the lowest concentration detected by 19 out of 20 replicates in an experiment so they used 20 replicates in attempts to detect 3.75 ng/mL nucleocapsid protein². 19 of the 20 replicates produced a positive result, establishing the new limit of detection for the prototype at 3.75 ng/mL.

Strategy 2: Increasing lateral flow assay sensitivity by increasing incubation time between conjugate and analyte

To further improve sensitivity of their lateral flow assay prototype, the researchers examined whether it would be possible to increase the contact between analyte and antibody. In theory, two approaches can accomplish this: (1) increasing the contact time between conjugate and antibody or (2) increasing colloidal gold conjugate concentration. Previous studies have placed porous materials between the conjugate pad and nitrocellulose membrane to increase the interaction time. For instance, adding a sponge decreases fluid flow rates leading to a 10-fold increase in signal detection in a nucleic acid hybridization-based lateral flow assay³. Since increasing the conjugate optical density is costly and is a time-consuming process, the researchers focused on increasing contact time between conjugate and antibody using porous materials.

First, the team added a 7 mm glass fiber or cotton fiber intermembrane between the conjugate pad and nitrocellulose membrane to their prototype. They found that adding a cotton intermembrane provided a similar sensitivity enhancement as increasing colloidal gold conjugate optical density. When tested at a nucleoprotein concentration at 2.0 ng/ml they found that 19 of the 20 replicates produced a positive signal, indicating a lower limit of detection with this further optimized prototype.

Limit of Detection of Lateral Flow Tests on the Market

For easily transmissible respiratory illnesses, it’s important to detect viruses early. This often means detecting a lower viral load, which requires rapid tests with more sensitivity. Many SARS-CoV-2 rapid tests on the market detect between 0.6 ng/mL and 10 ng/mL which puts the prototype described by the authors at the lower end of that range⁴.

Download the Lateral Flow Assay Development Guide

References

Nicollete DRP, Benedetti R, Valença BA, et al. Enhancing a SARS-CoV-2 Nucleocapsid Antigen Test Sensitivity with Cost Efficient Strategy through a Cotton Intermembrane Insertion. Sci Rep. 2023;13(1):4690.
FDA. Template for Developers of Antigen Tests. [Internet]. FDA; 2019. Available from: https://www.fda.gov/media/137907/download
Tang R, Yang H, Gong Y, et al. Improved Analytical Sensitivity of Lateral Flow Assay Using Sponge for HBV Nucleic Acid Detection. Sci Rep. 2017;7(1): 1360.
Corman VM, Haage VC, Bleicker T, et al. Comparison of Seven Commercial SARS-CoV-2 Rapid Point-of-Care Antigen Tests: A Single-Centre Laboratory Evaluation Study. The Lancet Microbe. 2021;2(7):e311–e319.