On the face of it a lateral flow assay or strip test is very simple; involving the chromatographic separation of a test solution across a nitrocellulose membrane and the identification of a specific analyte by binding to affinity reagents on the test strip to give a signal.
Evolution of the lateral flow assay format now means that results can be qualitative, semi-quantitative or quantitative and that multiplexing capabilities are available. Because of the combination of rapid results with cost effective point-of-care testing by non-specialists offered by the lateral flow assay platform it has been adopted across a variety of industries, from personal care products to environmental remediation and medical diagnostics, with the result that the global market for lateral flow assays now has an estimated value of $6.78 billion by 2020 (link is external).
Applications for the lateral flow assay, such as detection of cardiac biomarkers, biothreats and single cell signals, have driven developments in assay design. The inclusion of new materials and labelling systems, like quantum dots and carbon nanoparticles, have helped to increase sensitivity. Incorporating these technologies allows a fluorescent read-out that increases the sensitivity of the assay by several fold and can mean that in some cases they can be reused. One example of this is the development of a multiplexed lateral flow assay that can identify different foot-and-mouth disease serotypes (link is external) in cattle stock making treatment quicker and easier. What’s more in a world where nothing is more important to us than our phones, the fluorescent lateral flow assay has already been adapted for use in the field by using mobile phones to detect the signal.
Using up-converting phosphor technologies in lateral flow assays can give another 10-100 fold increase in sensitivity compared to fluorescence detection. This technology uses particles of rare earth elements embedded in crystal that can up-convert low-energy infrared light signals to high-energy visible light with red, green and blue emissions. Recent developments in lateral flow assays have used this technology to produce assays that include the capability to detect the biowarfare pathogen Francisella tularensis (link is external) and one that can determine the presence of ten different common food pathogens (link is external) within one sample in a multiplex format, among many others.
Despite these advances, colloidal gold is still the most commonly used label in lateral flow assays, offering the benefits of being inert, stable to heat and light, easy to visualise and easy to conjugate with biological materials, making assays easy to manufacture and store long-term.
For lateral flow assays to reach their full potential a number of issues need to be addressed- issues of bottlenecks in assay development and production, signal sensitivity and specificity, difficult assay targets and assay shelf-life and storage. We believe Affimer technology offers solutions to these challenges, increasing the potential of lateral flow diagnostics via a number of avenues.
All the materials making up a lateral flow assay, including colloidal gold, can be easily produced at low cost, making the production of specific affinity reagents the common bottleneck in assay development and production. The rapid production times for Affimer reagents offer a clear advantage here, our specific zika (link is external) virus binders were identified and characterised in just 13 weeks. What’s more as Affimer proteins lack complex post-translational modifications they can be produced in simple prokaryotic systems, meaning that scaling up of production is not as resource intensive.
One way improving sensitivity and target specificity has been addressed is to attempt to orient the detection reagents within the test strip to improve capture. Thiol chemistry can be easily used for specifically orienting Affimer reagents on solid supports while the small size of these affinity reagents allows increased surface density offering greater signal to noise ratio in the assay.
Sourcing binding reagents for toxic or non-immune targets, such as drugs, pesticides and chemical contaminants has prevented lateral flow assay development in these areas. But, Affimer proteins are identified from library screens and don’t rely on an immune system (link is external) for generation, so using Affimer technology these targets become accessible and potential for this rapid sensitive screening method to be employed in new areas opens up.
Affimer proteins are very robust and so resistant to a wide range of assay conditions, such as high temperatures and a wide pH range. The harsh chemical conditions found in test samples such as urine, stomach acid and environmental samples mean that complex pre-treatments are often used to make the samples compatible with the assay affinity reagents. Using Affimer proteins can negate these steps, offering quicker simpler tests.
Most lateral flow assays require specified temperatures for storage and have relatively short shelf-lives for optimal stability, while certain assays are entirely intolerant of higher temperatures. This is usually a function of the affinity reagent stability in the system and has a high cost impact on the supply chain and inhibits their use in some field environments. Affimer reagents show long shelf lives even at room temperatures and have been shown to be stable and remain functional to much higher temperatures, removing this obstacle to use and opening up convenient, point-of-care testing for use in a wider range of environments and across a greater range of analytes.
The engineered characteristics of Affimer reagents mean that they could increase the potential of lateral flow diagnostics by addressing the many different current drawbacks. For more information about Affimer reagents or to explore our custom Affimer services contact us (link is external).