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).

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

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.

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]

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 is the home pregnancy test.[2]

COVID-19 testing

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.[28] The systematic evaluation of lateral flow assays during the COVID-19 pandemic[29] 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.[30][31][32] 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[32] in viral antigen detection/sensitivity with excellent specificity, although kit failure rates and the impact of training were potential issues.[31] The Innova test's specificity is more widely publicised, but sensitivity in phase 4 trials was 50.1%.[33] 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.[34] Biweekly LFT were made universally available to everyone in England on April 9, 2021.[35] LFTs have been used for mass testing for COVID-19 globally[36][37][38] and complement other public health measures for COVID-19.[39]

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".[40][41]

Sensitivity of tests used in 2022 was around 70%.[42]

See also

References

  1. ↑ Weiss, Alan (1 November 1999). "Concurrent engineering for lateral-flow diagnostics". IVD Technology. Archived from the original on 2014-04-15. Retrieved 1 October 2022.
  2. 1 2 3 4 5 6 Koczula, Katarzyna M.; Gallotta, Andrea (30 June 2016). "Lateral flow assays". Essays in Biochemistry. 60 (1): 111–120. doi:10.1042/EBC20150012. PMC 4986465. PMID 27365041.
  3. ↑ Yetisen AK, Akram MS, Lowe CR (June 2013). "Paper-based microfluidic point-of-care diagnostic devices". Lab on a Chip. 13 (12): 2210–51. doi:10.1039/C3LC50169H. PMID 23652632. S2CID 17745196.
  4. ↑ "Lateral Flow Introduction". khufash.com. Archived from the original on 28 July 2012. Retrieved 27 July 2012.
  5. ↑ Hansson J, Yasuga H, Haraldsson T, van der Wijngaart W (January 2016). "Synthetic microfluidic paper: high surface area and high porosity polymer micropillar arrays". Lab on a Chip. 16 (2): 298–304. doi:10.1039/C5LC01318F. PMID 26646057.
  6. 1 2 Weijin Guo; Jonas Hansson; Wouter van der Wijngaart (2016). "Viscosity Independent Paper Microfluidic Imbibition" (PDF). MicroTAS 2016, Dublin, Ireland.
  7. ↑ Crozier, Alex; Rajan, Selina; Buchan, Iain; McKee, Martin (3 February 2021). "Put to the test: Use of rapid testing technologies for Covid-19". BMJ. 372: Appendix p. 6. doi:10.1136/bmj.n208. PMID 33536228. S2CID 231775752. Appendix Table 1, p. 6.
  8. ↑ "Antibodies, Proteins, ELISA kits". Abbexa. Retrieved 12 January 2022. Lateral Flow Assays, also known as Lateral Flow Immunochromatographic Assays [...] The technology is based on a series of capillary beds; such as pieces of porous paper, micro-structured polymer, or sintered polymer. Each of these elements has the capacity to transport fluid (e.g., urine), spontaneously.
  9. 1 2 Haslam E (2007). "Vegetable tannins - lessons of a phytochemical lifetime". Phytochemistry. 68 (22–24): 2713–21. Bibcode:2007PChem..68.2713H. doi:10.1016/j.phytochem.2007.09.009. PMID 18037145.
  10. ↑ Consden R, Gordon AH, Martin AJ (1944). "Qualitative analysis of proteins: a partition chromatographic method using paper". The Biochemical Journal. 38 (3): 224–32. doi:10.1042/bj0380224. PMC 1258072. PMID 16747784.
  11. ↑ "Paper chromatography | chemistry". Encyclopedia Britannica. Retrieved 2018-06-01.
  12. ↑ Engvall, E (1972-11-22). "Enzyme-linked immunosorbent assay, Elisa". The Journal of Immunology. 109 (1): 129–135. doi:10.4049/jimmunol.109.1.129. ISSN 0022-1767. PMID 4113792.
  13. 1 2 US patent 6485982, David E. Charlton, "Test device and method for colored particle immunoassay", published November 26, 2002, assigned to Church & Dwight
  14. ↑ Quesada-González D, Merkoçi A (November 2015). "Nanoparticle-based lateral flow biosensors". Biosensors & Bioelectronics. 73 (special): 47–63. doi:10.1016/j.bios.2015.05.050. hdl:10261/131760. PMID 26043315.
  15. ↑ NanoHybrids. "Gold Nanoparticle Labels Custom Designed for Lateral Flow Assays". NanoHybrids. Archived from the original on 11 September 2021. Retrieved 29 September 2020.
  16. ↑ Faulstich K, Gruler R, Eberhard M, Haberstroh K (July 2007). "Developing rapid mobile POC systems. Part 1: Devices and applications for lateral-flow immunodiagnostics". IVD Technology. 13 (6): 47–53. Archived from the original on 2009-03-02. Retrieved 2007-11-22.
  17. ↑ LaBorde RT, O'Farrell B (April 2002). "Paramagnetic-particle detection in lateral-flow assays". IVD Technol. 8 (3): 36–41. Archived from the original on 2010-03-01. Retrieved 2007-11-22.
  18. ↑ "Magnetic immunoassays: A new paradigm in POCT (IVDT archive, Jul/Aug 2008)". Archived from the original on 28 October 2013. Retrieved 2008-10-23.
  19. 1 2 3 nanoComposix. "Introduction to Lateral Flow Rapid Test Diagnostics". nanoComposix. Retrieved 4 November 2019.
  20. ↑ "Detekt Biomedical L.L.C.- Lateral Flow Readers for Rapid Test Strip Detection and Immunoassays". idetekt.com. Retrieved 6 July 2017.
  21. ↑ Guo, Weijin; Hansson, Jonas; Van Der Wijngaart, Wouter (2016). "Capillary Pumping Independent of Liquid Sample Viscosity". Langmuir. 32 (48): 12650–12655. doi:10.1021/acs.langmuir.6b03488. PMID 27798835. S2CID 24662688.
  22. ↑ Guo, Weijin; Hansson, Jonas; Van Der Wijngaart, Wouter (2017). "Capillary pumping with a constant flow rate independent of the liquid sample viscosity and surface energy". 2017 IEEE 30th International Conference on Micro Electro Mechanical Systems (MEMS). pp. 339–341. doi:10.1109/MEMSYS.2017.7863410. ISBN 978-1-5090-5078-9. S2CID 13219735.
  23. ↑ Guo W, Hansson J, van der Wijngaart W (2018). "Capillary pumping independent of the liquid surface energy and viscosity". Microsystems & Nanoengineering. 4 (1): 2. Bibcode:2018MicNa...4....2G. doi:10.1038/s41378-018-0002-9. PMC 6220164. PMID 31057892.
  24. ↑ Tripathi S, Kumar V, Prabhakar A, Joshi S, Agrawal A (2015). "Passive blood plasma separation at the microscale: a review of design principles and microdevices". J. Micromech. Microeng. 25 (8): 083001. Bibcode:2015JMiMi..25h3001T. doi:10.1088/0960-1317/25/8/083001. S2CID 138153068.
  25. ↑ Guo W, Hansson J, van der Wijngaart W (May 2020). "Synthetic Paper Separates Plasma from Whole Blood with Low Protein Loss". Analytical Chemistry. 92 (9): 6194–6199. doi:10.1021/acs.analchem.0c01474. PMID 32323979.
  26. ↑ Liu, Yilin; Zhan, Li; Qin, Zhenpeng; Sackrison, James; Bischof, John C. (23 March 2021). "Ultrasensitive and Highly Specific Lateral Flow Assays for Point-of-Care Diagnosis". ACS Nano. 15 (3): 3593–3611. doi:10.1021/acsnano.0c10035. ISSN 1936-0851. PMID 33607867. S2CID 231969545.
  27. ↑ "Grassroots Web group challenging lateral-flow patents". Medical DeviceLink. November 2000. Archived from the original on 8 July 2001.
  28. ↑ Guglielmi G (September 2020). "Fast coronavirus tests: what they can and can't do". Nature. 585 (7826): 496–498. Bibcode:2020Natur.585..496G. doi:10.1038/d41586-020-02661-2. PMID 32939084. S2CID 221768935.
  29. ↑ "Guidance: First wave of non-machine based lateral flow technology (LFT) assessment". GOV.UK. 11 January 2021. Retrieved 20 January 2021.
  30. ↑ "Oxford University and PHE confirm high-sensitivity of lateral flow tests". GOV.UK. 11 November 2020.
  31. 1 2 "FALCON — CONDOR Platform". www.condor-platform.org. Retrieved 25 November 2020.
  32. 1 2 Peto T (June 2021). "COVID-19: Rapid antigen detection for SARS-CoV-2 by lateral flow assay: A national systematic evaluation of sensitivity and specificity for mass-testing". eClinicalMedicine. 36: 100924. doi:10.1016/j.eclinm.2021.100924. PMC 8164528. PMID 34101770. S2CID 231609922.
  33. ↑ Wolf A, Hulmes J, Hopkins H (10 March 2021). "Lateral flow device specificity in phase 4 (post marketing) surveillance" (PDF). gov.uk. Archived (PDF) from the original on 2022-08-14.
  34. ↑ "Coronavirus (COVID-19) asymptomatic testing in schools and colleges". GOV.UK. Retrieved 2021-05-27.
  35. ↑ "Twice weekly rapid testing to be available to everyone in England". GOV.UK. 2021-04-05. Retrieved 2021-05-27.
  36. ↑ "Slovakia carries out Covid mass testing of two-thirds of population". The Guardian. Agence France-Presse. 2 November 2020. ISSN 0261-3077.
  37. ↑ Peter Littlejohns (6 November 2020). "The UK is trialling lateral flow testing for Covid-19 – how does it work?". NS Medical Devices.
  38. ↑ "Merthyr Tydfil County Borough to be first whole area testing pilot in Wales". GOV.WALES. 18 November 2020.
  39. ↑ "Population-wide testing of SARS-CoV-2: country experiences and potential approaches in the EU/EEA and the United Kingdom". European Centre for Disease Prevention and Control. 19 August 2020.
  40. ↑ Burke M (18 November 2020). "Scientists urge caution on use of lateral flow tests to screen for Covid-19". Chemistry World. Criticism of LFDs for Covid testing by several experts, with detailed numerical discussion.
  41. ↑ Deeks J, Raffle A, Gill M (12 January 2021). "Covid-19: government must urgently rethink lateral flow test roll out". The BMJ Opinion.
  42. ↑ Tapari A, Braliou GG, Papaefthimiou M, Mavriki H, Kontou PI, Nikolopoulos GK, Bagos PG (4 June 2022). "Performance of Antigen Detection Tests for SARS-CoV-2: A Systematic Review and Meta-Analysis". Diagnostics. 12 (6): 1388. doi:10.3390/diagnostics12061388. PMC 9221910. PMID 35741198.

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