Lateral flow test

Last updated
A NASA illustration of a lateral flow assay Lateral Flow Assay.jpg
A NASA illustration of a lateral flow assay

A lateral flow test (LFT), [1] is an assay also known as a lateral flow device (LFD), lateral flow immunochromatographic assay, or rapid test. It is a simple device intended to detect the presence of a target substance in a liquid sample without the need for specialized and costly equipment. LFTs are widely used in medical diagnostics in the home, at the point of care, and in the laboratory. For instance, the home pregnancy test is an LFT that detects a specific hormone. These tests are simple and economical and generally show results in around five to thirty minutes. [2] Many lab-based applications increase the sensitivity of simple LFTs by employing additional dedicated equipment. [3] Because the target substance is often a biological antigen, many lateral flow tests are rapid antigen tests (RAT or ART).

Contents

LFTs operate on the same principles of affinity chromatography as the enzyme-linked immunosorbent assays (ELISA). In essence, these tests run the liquid sample along the surface of a pad with reactive molecules that show a visual positive or negative result. The pads are based on a series of capillary beds, such as pieces of porous paper, [4] microstructured polymer, [5] [6] or sintered polymer. [7] Each of these pads has the capacity to transport fluid (e.g., urine, blood, saliva) spontaneously. [8]

The sample pad acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid flows to the second conjugate pad in which the manufacturer has stored freeze dried bio-active particles called conjugates (see below) in a salt–sugar matrix. The conjugate pad contains all the reagents required for 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. This marks target particles as they pass through the pad and continue across to the test and control lines. The test line shows a signal, often a color as in pregnancy tests. The control line contains affinity ligands which show whether the sample has flowed through and the bio-molecules in the conjugate pad are active. After passing these reaction zones, the fluid enters the final porous material, the wick, that simply acts as a waste container.

LFTs can operate as either competitive or sandwich assays.

History

LFTs derive from paper chromatography, which was developed in 1943 by Martin and Synge, [9] and elaborated in 1944 by Consden, Gordon and Martin. [10] [11] There was an explosion of activity in this field after 1945. [9] The ELISA technology was developed in 1971. [12] A set of LFT patents, including the litigated US 6,485,982 described below, were filed by Armkel LLC starting in 1988. [13]

Synopsis

Colored particles

In principle, any colored particle can be used, but latex (blue color) or nanometer-sized particles [14] of gold (red color) are most commonly used. The gold particles are red in color due to localized surface plasmon resonance. [15] Fluorescent [16] or magnetic [17] [18] labelled particles can also be used, but these require the use of an electronic reader to assess the test result.

Sandwich assays

Difference between sandwich assay and competitive assay formats of lateral flow tests ELISA or Lateral flow formats.svg
Difference between sandwich assay and competitive assay formats of lateral flow tests

Sandwich assays are generally used for larger analytes because they tend to have multiple binding sites. [19] As the sample migrates through the assay it first encounters a conjugate, which is an antibody specific to the target analyte labelled with a visual tag, usually colloidal gold. The antibodies bind to the target analyte within the sample and migrate together until they reach the test line. The test line also contains immobilized antibodies specific to the target analyte, which bind to the migrated analyte bound conjugate molecules. The test line then presents a visual change due to the concentrated visual tag, hence confirming the presence of the target molecules. The majority of sandwich assays also have a control line which will appear whether or not the target analyte is present to ensure proper function of the lateral flow pad. [2]

The rapid, low-cost sandwich-based assay is commonly used for home pregnancy tests which detect human chorionic gonadotropin, hCG, in the urine of pregnant women.

Competitive assays

Competitive assays are generally used for smaller analytes since smaller analytes have fewer binding sites. [19] The sample first encounters antibodies to the target analyte labelled with a visual tag (colored particles). The test line contains the target analyte fixed to the surface. When the target analyte is absent from the sample, unbound antibody will bind to these fixed analyte molecules, meaning that a visual marker will show. Conversely, when the target analyte is present in the sample, it binds to the antibodies to prevent them binding to the fixed analyte in the test line, and thus no visual marker shows. This differs from sandwich assays in that no band means the analyte is present. [2] [19]

Quantitative tests

Most LFTs 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. [20] Alternative non-optical techniques are also able to report quantitative assays results. One such example is a magnetic immunoassay (MIA) in the LFT 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. [6] [21] [22] [23]

Control line

The control line of this pregnancy test is blank, making the test invalid. Negative Pregnancy Test.JPG
The control line of this pregnancy test is blank, making the test invalid.

Most tests will incorporate a second line which contains a further antibody (one which is not specific to the analyte) that binds some of the remaining colored particles which did not bind to the test line. This confirms that fluid has passed successfully from the sample-application pad, past the test line. [2] By giving confirmation that the sample has had a chance to interact with the test line, this increases confidence that a visibly-unchanged test line can be interpreted as a negative result (or that a changed test line can be interpreted as a negative result in a competitive assay).

Blood plasma extraction

Because the intense red color of hemoglobin interferes with the readout of colorimetric or optical detection-based diagnostic tests, blood plasma separation is a common first step to increase diagnostic test accuracy. Plasma can be extracted from whole blood via integrated filters [24] or via agglutination. [25]

Speed and simplicity

Time to obtain the test result is a key driver for these products. Tests results can be available in as little as a few minutes. Generally there is a trade off between time and sensitivity: 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, by typically requiring little or no sample or reagent preparation. [26]

Patents

This is a highly competitive area and a number of people claim patents in the field, most notably Alere (formerly Inverness Medical Innovations, now owned by Abbott) who own patents [13] originally filed by Unipath. The US 6,485,982 patent, that has been litigated, expired in 2019. A number of other companies also hold patents in this arena. A group of competitors are challenging the validity of the patents. [27] The original patent is apparently from 1988. [28] [29]

Applications

Lateral flow assays have a wide array of applications and can test a variety of samples like urine, blood, saliva, sweat, serum, and other fluids. They are currently used by clinical laboratories, hospitals, and physicians for quick and accurate tests for specific target molecules and gene expression. Other uses for lateral flow assays are food and environmental safety and veterinary medicine for chemicals such as diseases and toxins. [2] LFTs are also commonly used for disease identification such as ebola, but the most common LFT are the home pregnancy [2] and SARS-CoV-2 tests.

COVID-19 testing

COVID-19 rapid antigen lateral flow test showing a negative result Test de antigenos Covid 3.jpg
COVID-19 rapid antigen lateral flow test showing a negative result

Lateral flow assays have played a critical role in COVID-19 testing as they have the benefit of delivering a result in 15–30 minutes. [30] The systematic evaluation of lateral flow assays during the COVID-19 pandemic [31] was initiated at Oxford University as part of a UK collaboration with Public Health England. A study that started in June 2020 in the United Kingdom, FALCON-C19, confirmed the sensitivity of some lateral flow devices (LFDs) in this setting. [32] [33] [34] Four out of 64 LFDs tested had desirable performance characteristics according to these early tests; the Innova SARS-CoV-2 Antigen Rapid Qualitative Test performed moderately [34] in viral antigen detection/sensitivity with excellent specificity, although kit failure rates and the impact of training were potential issues. [33] The Innova test's specificity is more widely publicised, but sensitivity in phase 4 trials was 50.1%. [35] This describes a device for which one out of every two patients infected with COVID-19 and tested in real-world conditions would receive a false-negative result. After closure of schools in January 2021, biweekly LFTs were introduced in England for teachers, pupils, and households of pupils when schools re-opened on March 8, 2021 for asymptomatic testing. [36] Biweekly LFT were made universally available to everyone in England on April 9, 2021. [37] LFTs have been used for mass testing for COVID-19 globally [38] [39] [40] and complement other public health measures for COVID-19. [41]

Some scientists outside government expressed serious misgivings in late 2020 about the use of Innova LFDs for screening for Covid. According to Jon Deeks, a professor of biostatistics at the University of Birmingham, England, the Innova test is "entirely unsuitable" for community testing: "as the test may miss up to half of cases, a negative test result indicates a reduced risk of Covid, but does not exclude Covid". [42] [43]

Sensitivity of tests used in 2022 was around 70%. [44]

See also

Related Research Articles

<span class="mw-page-title-main">ELISA</span> Method to detect an antigen using an antibody and enzyme

The enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay is a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand in a liquid sample using antibodies directed against the ligand to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.

A biosensor is an analytical device, used for the detection of a chemical substance, that combines a biological component with a physicochemical detector. The sensitive biological element, e.g. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc., is a biologically derived material or biomimetic component that interacts with, binds with, or recognizes the analyte under study. The biologically sensitive elements can also be created by biological engineering. The transducer or the detector element, which transforms one signal into another one, works in a physicochemical way: optical, piezoelectric, electrochemical, electrochemiluminescence etc., resulting from the interaction of the analyte with the biological element, to easily measure and quantify. The biosensor reader device connects with the associated electronics or signal processors that are primarily responsible for the display of the results in a user-friendly way. This sometimes accounts for the most expensive part of the sensor device, however it is possible to generate a user friendly display that includes transducer and sensitive element. The readers are usually custom-designed and manufactured to suit the different working principles of biosensors.

An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity. The measured entity is often called the analyte, the measurand, or the target of the assay. The analyte can be a drug, biochemical substance, chemical element or compound, or cell in an organism or organic sample. An assay usually aims to measure an analyte's intensive property and express it in the relevant measurement unit.

<span class="mw-page-title-main">Immunoassay</span> Biochemical test for a protein or other molecule using an antibody

An immunoassay (IA) is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes). The molecule detected by the immunoassay is often referred to as an "analyte" and is in many cases a protein, although it may be other kinds of molecules, of different sizes and types, as long as the proper antibodies that have the required properties for the assay are developed. Analytes in biological liquids such as serum or urine are frequently measured using immunoassays for medical and research purposes.

<span class="mw-page-title-main">Hook effect</span> Immunologic phenomenon occurring in high antigen or antibody levels

The hook effect refers to the prozone phenomenon, also known as antibody excess, or the postzone phenomenon, also known as antigen excess. It is an immunologic phenomenon whereby the effectiveness of antibodies to form immune complexes can be impaired when concentrations of an antibody or an antigen are very high. The formation of immune complexes stops increasing with greater concentrations and then decreases at extremely high concentrations, producing a hook shape on a graph of measurements. An important practical relevance of the phenomenon is as a type of interference that plagues certain immunoassays and nephelometric assays, resulting in false negatives or inaccurately low results. Other common forms of interference include antibody interference, cross-reactivity and signal interference. The phenomenon is caused by very high concentrations of a particular analyte or antibody and is most prevalent in one-step (sandwich) immunoassays.

Heterophile antibodies are antibodies induced by external antigens that may be shared between species and are not well defined. They often have weak avidity for their targets.

Cross-reactivity, in a general sense, is the reactivity of an observed agent which initiates reactions outside the main reaction expected. This has implications for any kind of test or assay, including diagnostic tests in medicine, and can be a cause of false positives. In immunology, the definition of cross-reactivity refers specifically to the reaction of the immune system to antigens. There can be cross-reactivity between the immune system and the antigens of two different pathogens, or between one pathogen and proteins on non-pathogens, which in some cases can be the cause of allergies.

<span class="mw-page-title-main">Antibody microarray</span> Form of protein microarray

An antibody microarray is a specific form of protein microarray. In this technology, a collection of captured antibodies are spotted and fixed on a solid surface such as glass, plastic, membrane, or silicon chip, and the interaction between the antibody and its target antigen is detected. Antibody microarrays are often used for detecting protein expression from various biofluids including serum, plasma and cell or tissue lysates. Antibody arrays may be used for both basic research and medical and diagnostic applications.

<span class="mw-page-title-main">Rapid strep test</span> Test for strep throat

The rapid strep test (RST) is a rapid antigen detection test (RADT) that is widely used in clinics to assist in the diagnosis of bacterial pharyngitis caused by group A streptococci (GAS), sometimes termed strep throat. There are currently several types of rapid strep test in use, each employing a distinct technology. However, they all work by detecting the presence of GAS in the throat of a person by responding to GAS-specific antigens on a throat swab.

Magnetic immunoassay (MIA) is a type of diagnostic immunoassay using magnetic beads as labels in lieu of conventional enzymes (ELISA), radioisotopes (RIA) or fluorescent moieties to detect a specified analyte. MIA involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to one element of the pair. The presence of magnetic beads is then detected by a magnetic reader (magnetometer) which measures the magnetic field change induced by the beads. The signal measured by the magnetometer is proportional to the analyte concentration in the initial sample.

<span class="mw-page-title-main">Centrifugal micro-fluidic biochip</span>

The centrifugal micro-fluidic biochip or centrifugal micro-fluidic biodisk is a type of lab-on-a-chip technology, also known as lab-on-a-disc, that can be used to integrate processes such as separating, mixing, reaction and detecting molecules of nano-size in a single piece of platform, including a compact disk or DVD. This type of micro-fluidic biochip is based upon the principle of microfluidics to take advantage of non-inertial pumping; for lab-on-a-chip devices using non-inertial valves and switches under centrifugal force and Coriolis effect, this is in order to distribute fluids about the disks in a highly parallel order.

<span class="mw-page-title-main">Surround optical-fiber immunoassay</span>

Surround optical-fiber immunoassay (SOFIA) is an ultrasensitive, in vitro diagnostic platform incorporating a surround optical-fiber assembly that captures fluorescence emissions from an entire sample. The technology's defining characteristics are its extremely high limit of detection, sensitivity, and dynamic range. SOFIA's sensitivity is measured at the attogram level (10−18 g), making it about one billion times more sensitive than conventional diagnostic techniques. Based on its enhanced dynamic range, SOFIA is able to discriminate levels of analyte in a sample over 10 orders of magnitude, facilitating accurate titering.

Flow-through tests or immunoconcentration assays are a type of diagnostic assay that allows users to test for the presence of a biomarker, usually a specific antibody, in a sample such as blood. They are a type of point of care test, designed to be used by a healthcare provider at patient contact. Point of care tests often allow for rapid detection of a specific biomarker without specialized lab equipment and training; this aids in diagnosis and allows therapeutic action to be initiated more quickly. Flow-through tests began development in the early 1980s and were the first type of immunostrip to be developed, although lateral flow tests have subsequently become the dominant immunostrip point of care device.

Mass spectrometric immunoassay (MSIA) is a rapid method is used to detect and/ or quantify antigens and or antibody analytes. This method uses an analyte affinity isolation to extract targeted molecules and internal standards from biological fluid in preparation for matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). This method allows for "top down" and "bottom up" analysis. This sensitive method allows for a new and improved process for detecting multiple antigens and antibodies in a single assay. This assay is also capable of distinguishing mass shifted forms of the same molecule via a panantibody, as well as distinguish point mutations in proteins. Each specific form is detected uniquely based on their characteristic molecular mass. MSIA has dual specificity because of the antibody-antigen reaction coupled with the power of a mass spectrometer.

<span class="mw-page-title-main">Rapid diagnostic test</span>

A rapid diagnostic test (RDT) is a medical diagnostic test that is quick and easy to perform. RDTs are suitable for preliminary or emergency medical screening and for use in medical facilities with limited resources. They also allow point-of-care testing in primary care for things that formerly only a laboratory test could measure. They provide same-day results within two hours, typically in approximately 20 minutes.

<span class="mw-page-title-main">Rapid antigen test</span> Fast medical lateral flow test

A rapid antigen test (RAT), sometimes called a rapid antigen detection test (RADT), antigen rapid test (ART), or loosely just a rapid test, is a rapid diagnostic test suitable for point-of-care testing that directly detects the presence or absence of an antigen. RATs are a type of lateral flow test detecting antigens, rather than antibodies or nucleic acid. Rapid tests generally give a result in 5 to 30 minutes, require minimal training or infrastructure, and have significant cost advantages. Rapid antigen tests for the detection of SARS-CoV-2, the virus that causes COVID-19, have been commonly used during the COVID-19 pandemic.

<span class="mw-page-title-main">Fluorescence polarization immunoassay</span> Class of invitro biochemical test

Fluorescence polarization immunoassay (FPIA) is a class of in vitro biochemical test used for rapid detection of antibody or antigen in sample. FPIA is a competitive homogenous assay, that consists of a simple prepare and read method, without the requirement of separation or washing steps.

<span class="mw-page-title-main">Multiplexed point-of-care testing</span> Bedside testing technology

Multiplexed point-of-care testing (xPOCT) is a more complex form of point-of-care testing (POCT), or bedside testing. Point-of-care testing is designed to provide diagnostic tests at or near the time and place that the patient is admitted. POCT uses the concentrations of analytes to provide the user with information on the physiological state of the patient. An analyte is a substance, chemical or biological, that is being analyzed using a certain instrument. While point-of-care testing is the quantification of one analyte from one in vitro sample, multiplexed point-of-care testing is the simultaneous on-site quantification of various analytes from a single sample.

Abingdon Health is a British manufacturer of lateral flow assay diagnostic tests, sometimes called rapid tests, lateral flow immunoassays (LFIA), lateral flow tests (LFT) or quick tests. Since its formation in 2008, Abingdon Health has developed and manufactured lateral flow rapid tests across multiple industries. Headquartered in York, UK, with an additional office in Doncaster, UK.

<span class="mw-page-title-main">COVID-19 rapid antigen test</span> Diagnostic test for a SARS-CoV-2 infection

COVID-19 rapid antigen tests or RATs, also frequently called COVID-19 lateral flow tests or LFTs, are rapid antigen tests used to detect SARS-CoV-2 infection (COVID-19). They are quick to implement with minimal training, cost a fraction of other forms of COVID-19 testing, and give users a result within 5–30 minutes. RATs have been used in several countries as part of mass testing or population-wide screening approaches. Many RATs can be used for self-testing, in which an individual "collects their own specimen… and interpret[s] their test result themselves".

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