Lateral flow test
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Lateral flow tests, also known as lateral flow immunochromatographic assays, are simple paper-based devices intended to detect the presence (or absence) of a target analyte in liquid sample (matrix) without the need for specialized and costly equipment, though many lab-based applications exist that are supported by reading equipment. Typically, these tests are used for medical diagnostics either for home testing, point of care testing, or laboratory use. A widely spread and well known application is the home pregnancy test.
The technology is based on a series of capillary beds, such as pieces of porous paper, microstructured polymer, or sintered polymer. Each of these elements has the capacity to transport fluid (e.g., urine) spontaneously.
The first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element (conjugate pad) in which the manufacturer has stored the so-called conjugate, a dried format of bio-active particles (see below) in a salt-sugar matrix that contains everything to guarantee an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle's surface. While the sample fluid dissolves the salt-sugar matrix, it also dissolves the particles, and in one combined transport action, the sample and conjugate mix while flowing through the porous structure. In this way, the analyte binds to the particles while migrating further through the third capillary bed. This material has one or more areas (often called stripes) where a third molecule has been immobilized by the manufacturer. By the time the sample-conjugate mix reaches these strips, analyte has been bound on the particle and the third "capture" molecule binds the complex. After a while, when more and more fluid has passed the stripes, particles accumulate and the stripe-area changes color. Typically, there are at least two stripes: one (the control) that captures any particle and thereby shows that reaction conditions and technology worked fine and one that contains a specific capture molecule and only captures those particles onto which an analyte molecule has been immobilized. After passing these reaction zones, the fluid enters the final porous material, the wick, that simply acts as a waste container.
In principle, any coloured particle can be used, however latex (blue colour) or nanometer sized particles of gold (red colour) are most commonly used. The gold particles are red in colour due to localised surface plasmon resonance. Fluorescent or magnetic labeled particles can also be used, however these require the use of an electronic reader to assess the test result.
As the sample migrates along the assay it first encounters a conjugate, usually colloidal gold, which is labelled with antibodies specific to the target analyte. If the target analyte is detected within the sample the conjugate antibodies will bind and subsequently reach the test line which also contains antibodies specific to the target. Once the sample reaches the test line and the target analyte is present a visual change, normally a line appearing, will occur allowing the test to be read as a positive. The majority of sandwich assays also have a control line which will appear regardless of whether or not the target analyte is present.
The rapid, low-cost sandwich-based assay is commonly used for home pregnancy tests which detects for human chorionic gonadotropin, hCG, in the urine of women.
The sample first encounters coloured particles which are labelled with the target analyte or an analogue. The test line contains antibodies to the target/its analogue. Unlabeled analyte in the sample will block the binding sites on the antibodies preventing uptake of the coloured particles.
The test line will show as a coloured band in negative samples.
Most tests are intended to operate on a purely qualitative basis. However it is possible to measure the intensity of the test line to determine the quantity of analyte in the sample. Handheld diagnostic devices known as lateral flow readers are used by several companies to provide a fully quantitative assay result. By utilizing unique wavelengths of light for illumination in conjunction with either CMOS or CCD detection technology, a signal rich image can be produced of the actual test lines. Using image processing algorithms specifically designed for a particular test type and medium, line intensities can then be correlated with analyte concentrations. One such handheld lateral flow device platform is made by Detekt Biomedical L.L.C.. Alternative non-optical techniques are also able to report quantitative assays results. One such example is a magnetic immunoassay (MIA) in the lateral flow test form also allows for getting a quantified result. Reducing variations in the capillary pumping of the sample fluid is another approach to move from qualitative to quantitative results. Recent work has, for example, demonstrated capillary pumping with a constant flow rate independent from the liquid viscosity and surface energy.
Mobile phones have demonstrated to have a strong potential for the quantification in lateral flow assays, not only by using the camera of the device, but also the light sensor or the energy supplied by the mobile phone battery.
While not strictly necessary, most tests will incorporate a second line which contains an antibody that picks up free latex/gold in order to confirm the test has operated correctly.
Speed and simplicity
Time to obtain the test result is a key driver for these products. Tests can take as little as a few minutes to develop. Generally there is a trade off between time and sensitivity – so more sensitive tests may take longer to develop. The other key advantage of this format of test compared to other immunoassays is the simplicity of the test – typically requiring little or no sample or reagent preparation.
This is a highly competitive area and a number of people claim patents in the field, most notably Alere who own patents originally filed by Unipath. A group of competitors to Inverness Medical Innovations are challenging the validity of the patents. A number of other companies also hold patents in this arena.
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