Contributed Commentary by Morgan West and Ffion Walters
December 12, 2017 | Lateral flow immunoassays (LFIs) are simple yet powerful diagnostic tools, offering user-friendly, reliable, and low-cost testing. LFIs provide fast qualitative results, and are increasingly used in point‑of-care situations, without the need for laboratories or trained analysts. Most people are probably familiar with LFIs in the form of home pregnancy kits that detect human chorionic gonadotropin (hCG), a hormone released in the early stages of pregnancy that inhibits the shedding of the uterus lining. However, LFIs are also used to detect drugs, pesticides, pathogens and metal ion contaminants, leading to applications in environmental testing, medical and veterinary healthcare, and food and beverage quality assurance.
LFIs rely on the specific interaction between the target analyte (e.g. hormone, antibody or antigen) and a binding partner (e.g. antibody, antigen or enzyme), conjugated to a reporter label (often a gold nanoparticle). The complex of target analyte, binding partner, and reporter label is drawn through a membrane via capillary action towards a test line of further antibodies. The target analyte in the complex binds to the test line antibodies, immobilizing the complex, which we visually observe as a colored line indicating the presence of the target analyte.
A goal in LFI development is increasing signal intensity to produce less ambiguous results; it’s not uncommon to conduct tests in a lab where the signal is visible to trained clinical experts, but may be misinterpreted by less experienced end users in an everyday setting. With improved signal intensity comes the added benefit that it takes less time for a positive signal to appear, and a shorter time to result has obvious benefits in applications such as emergency medicine, where faster diagnosis can have life-saving implications.
Alongside higher signal intensity, scientists are also seeking to increase assay sensitivity and improve the limit of detection—the lowest analyte concentration that can be distinguished within an assay. For example, increased levels of Troponin I in the blood indicate damage to the heart, and can be used to confirm a suspected acute myocardial infarction. Improving the limit of detection enables lower levels of Troponin I to be detected, allowing heart damage to be addressed earlier.
A first step towards increasing LFI sensitivity is developing high quality gold nanoparticles for diagnostic purposes. Poorly controlled manufacturing processes produce irregular particles prone to clustering, causing uneven surface area, inconsistent conjugation of the binding partner, and impeded movement through the test membranes, all of which lead to an ill-defined signal that is open to misinterpretation. A high level of reproducibility in terms of the size dispersion and shape is essential for the development of sensitive and reliable LFIs with minimal lot-to-lot variation.
However, improving gold quality can only take us so far towards increasing sensitivity. During manufacture of these tests, conjugation of the binding partner to the reporter label does not achieve complete saturation, resulting in “free” sites on the gold nanoparticles. To avoid non-specific binding, which can lead to cross-reactions and unwanted signals, a “conjugate blocking” agent is used. Bovine serum albumin (BSA) – a protein derived from cows – is the industry standard used in 95% of LFIs, but in solving one problem, it creates a few more.
BSA reacts with human serum albumin-binding antibodies, as well as bovine- and other animal-origin analytes, and it is difficult to minimise lot-to-lot variability in the manufacturing of the assays. The relatively large size of the BSA molecule (67 kDa) can also sterically hinder the target analyte from the binding partner, leading to reduced sensitivity. While several other alternatives are available – such as dry milk proteins, polyethene glycol and fish gelatin – these still suffer from lot-to-lot variability and non-specific binding issues.
As LFIs continue to establish themselves at the forefront of point-of-care diagnostics, expect to see more innovative solutions, propelled by the growing demand for better reproducibility, increased sensitivity and a shorter time to assay results.
Morgan West is New Product Development Leader with a degree in biochemistry. He has more than seven years of experience developing a wide range of clinical lateral flow assays, as well as infectious disease and veterinary tests. He can be reached at MorganWest@bbisolutions.com.
Ffion Walters is Senior Conjugation Scientist with a degree in Forensic Science and Masters in Renewable Energy. He has more than eight years of experience developing and manufacturing an array of nanoparticle based conjugations utilised in lateral flow assays. He can be reached at FfionWalters@bbisolutions.com.