Application Of Lateral Flow Immunoassay Biology Essay

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The present study conducted to understand the literatures related with the development, types and application of Lateral Flow Immunoassay. Nowadays we are frequently doing some diagnosis of disease, food health test, very sensitive issue like pregnancy test, just inside home within couple of minutes by a single small sized strip; actually that is 'Lateral Flow Immunoassay' (LFIA). Sometimes, this test incorrectly referred to as 'dipsticks' and ELISA (Enzyme Linked Immunosorbent Assay). The main principle of LFIA is different from the 'dipstick' where no 'lateral flow' occurred like LFIA though 'dipstick' has a membrane. On the other hand, there is a vast similarity in the basic principles of ELISA and LFIA except ELISA has no membrane like LFIA on which sample can flow. The basic working principle of LFIA is the movement of liquid sample containing target analyte just on a zoned porous membrane thereby passing different zones for the analyte-antibody interaction (Posthuma-Trumpie et al., 2009). This literature review discusses about concerning format, operation, range and types with application of LFIA.

The milestone research titled 'The Latex Fixation Test' in 1956 (Plotz and Singer, 1956), the Nobel prize winning invention 'Radio Immunoassay (RIA)'in 1959 (Berson and Yallow, 1959) and the development of 'radioimmunoassay for measurement of human chorionic gonadotropin (hCG)' in 1972 (Vaitukaitis et al., 1972) paved the way of the massive development of LFIA as we are using in present days.

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Figure-1: Typical configuration of a lateral flow immunoassay test strip. Figure copied and pasted from http://www.indicia.fr/pages/en/8/rapid-tests.html.

A typical LFIA format consists of a plastic backing with an adhesive, a sample pad, a nitrocellulose membrane (migration membrane), an absorbent pad and printed tapes for identification (Figure-1). Sample to be tested added to the sample pad and then it goes to conjugate pad (a site of immobilized particulate conjugate). Herein, the conjugate may be either antigen or antibody that must depend on the objective and format of the test. Then the dried conjugate get re-mobilizes by the sample and the analyte interacts with the conjugate. After that they migrate to the membrane which is also called 'the reaction matrix'. Reaction matrix is basically porous membrane consists of other specific immobilized components of the assay. Those components are either antibody or antigen and present in test line or control line according to the test objectives. Test line and control line acts as the capture zone of the analyte when sample passes that lines. Extra sample that passed the test line and control line go straight to the wick or the absorbent pad. Visible colour forms in control line and or test line which indicate the test results (O'Farrell, 2009 cited in Wong and Tsc, 2009). The distance between the conjugation pad and test line is important for the optimization of the efficiency of the assay. In some cases extra time gained for the distant position of the test line which allows more interaction (Posthuma-Trumpie et al., 2009).

The sample pad is the first entry point of the sample mostly made of cellulose or cross-linked silica. The membrane is must be thin and porous attached on a plastic base. Its robustness is gained by hosting strips in a plastic holder. The membrane strips are usually made up of nitrocellulose, nylon, polyether-sulfone, polyethylene or fused silica depending on the test purpose. In between the sample pad and the membrane, a conjugate pad is present mostly made up of cross-linked silica. Dried form of labelled analyte or recognition element(s) is laid in this pad. Coloured or fluorescent nanoparticles are the main constituents of labels. Particle may be gold nanoparticle, fluorescent, paramagnetic monodisperse latex particle and or recently getting popular quantum dots or up-converting phosphor. The size of the nanoparticle is between 15nm and 800nm. (Posthuma-Trumpie et al., 2009).

Gold nanoparticles are vastly using in lateral flow immunoassay for its 3 distinct properties such as low toxicity, high extinction coefficients and straight forward synthesis methods (Bailes et al., 2012). Latex is another popular one. Selenium, carbon and liposome are also used but fewer than gold and latex. Recently, quantum dots and up-converting phosphor are also using as label. A new technology has been developed called 'Up-Converting Phosphor Technology' (UPT) for LFIA based on lanthanide-bearing sub-micrometer-sized ceramic particles which can absorb infrared light but also emit visible light. UTP technology can be use in both 'sandwich' and 'competitive assay'. UTP technology based LFIA has opened a new door of LFIA application for on-site immunoassay with sensitivity which is absent in other label technology (Niedbala et al., 2001).

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Detection limit is a important factor of LFIA. There are some factors which are responsible for the detection limit of LFIA such as the size of gold nanoparticles, the antibody concentration, the conjugation pH, and characteristics of reaction matrix (Safenkova et al., 2012). If the reaction limit is lesser, then the efficiency of the particular LFIA will be improved. A LFIA strip developed using gold nanoparticle (AuNP) doubly labelled complex, in which biotinylated single-stranded DNA was used as a linkage to integrate 2 AuNPs and streptavidin-labeled AuNP, as an amplifier to magnify extremely low signals which can detect cardiac myoglobin and troponin with the detection limit of only 1 ng/L that is 1000 times lower than the generally used LFIA method for the same detection (Zhu et al., 2011).

Evaluation of three commercially important frequently used immunodiagnostic devices (enzyme-linked immunosorbent assays, lateral flow device and sandwich ELISA) for the qualitative and quantitative detection of three toxins (ricin, amanitin and T-2 toxin) worked out successfully in food with oral LD50 values > or = 0.1 mg/kg of body weight. Evaluation research concluded that Lateral flow devices and sandwich ELISA showed better performance than competitive ELISA (Garber et al., 2005).

An LFIA method is of two types, first, 'the competitive method', which is compatible for low molecular mass analyte showing single antigenic determinant. Second, 'the sandwich method' suitable for comparatively higher molecular mass analyte which have at least two epitopes and deploy two different antibody i.e. monoclonal (at the test line that plays detecting role) and polyclonal (at the conjugate pad that plays capturing role) (Ngom et al., 2010).

In competitive format of LFIA an analyte-protein conjugate present in the test zone of the nitrocellulose membrane catches a labelled anti-analyte monoclonal antibody complex. As a result colour particle get chance to produce visible sign on the test zone. On the other hand, another specific antibody from the control line catches the excess antibody complex which also produces visible line on the control zone whether the target analyte is present in the sample or not. So, for the competitive format, negative sample shows two visible colour bands (one in control line another in test line) (Ngom et al., 2010).

By using competitive format a number of test strips have been developed for various purposes. The materials used in those strip are different due to their use. Not only that, all strips are varies from their test time, detection limit, sensitivity and application. As for example, a competitive LFIA strip for the detection of the major peanut allergen has been developed. Devoid of the need of sophisticated kit or methods that strip gave results within 30 minutes and also cost-effective, quick, simple, and sensitive method for on-site detection of peanut allergens (Wen et al., 2005). Likewise, Nitrofuran family of antibiotics are used to protect cattle from disease as well as growth inducer which is a great threat in the food industry based on animal and a big issue for the health concerns nowadays. a LFIA strip outlined, anchored in competitive format to detect '1-aminohydantoin (AHD)' (a member of nitrofuran antibiotic family) in meat samples which is also quick and simple (Tang et al., 2011).

In competitive format, most of the cases researchers use gold nanoparticles. Whereas, others particle like carbon is also using recently. For instance, a competitive two steps lateral flow immunoassay based on carbon nanoparticles to determinate phytoregulator forchlorfenuron in horticultural sample has been prepared(Suárez-Pantaleón et al., 2013). Likewise, gold nanoparticles are also challenged by chemiluminescence. Research claimed that a biosensor of competitive lateral flow immunoassay based on enzyme-catalyzed chemiluminescence is much better than that of conventional LFIA based on colloidal gold particle to detect fumonisins (fumonisin B1+fumonisin B2) in maize (Mirasoli et al., 2012).

However, gold nanopariticle based competitive LFIA may give qualitative, semi-quantitative and even quantitative results. A lateral flow test strip in competitive format has been constructed contains gold-conjugated monoclonal antibody to determine the CL residues which was very useful for the quantitative, semi-quantitative as well as qualitative detection of Clenbuterol (CL) residues in swine urine (Zhang et al., 2006).

Sandwich format involves two different antibodies, one polyclonal and another monoclonal that bound specific epitopes of the analyte. In the conjugate pad dried form of a labelled polyclonal antibody remains coated and acts as a detector reagent. On the contrary, a monoclonal antibody which is specific to the analyte is placed at the test line of the membrane and acts as a capture reagent. When the sample migrates by capillary force to the membrane through the conjugate pad then the detector reagent released. A portion of analyte caught by the detection antibody and remain portion go ahead and passes through the capture zone. At this time both bound and unbound analytes bind to the capture antibody and produce signal (Ngom et al., 2010).

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In sandwich format, just like competitive format gold nanoparticles are mostly using but recent research suggests that quantum dot could be the efficient one than gold nanoparticles. As for example, to detect avian influenza virus (AIV) by traditional sandwich LFIA method the gold nanoparticle labels were using frequently. However, using label-free quantum dot in sandwich LFIA can give more efficiency (90.0%), sensitivity (100.0%) and specificity (88.2%) than gold particles (Li et al., 2012). Additionally, 'up-converting phosphor technology' also gives good efficiency in test. To detect plague disease caused by Yersinia pestis up-converting phosphor technology has been used successfully in sandwich format (Hong et al., 2010).

Sandwich LFIA may be 'Double Antibody Sandwich LFIA' (DAS-LFIA). In this case hepatitis C virus (HCV) detection is possible where strip developed by a sequence of various recombinant HCV proteins in Escherichia coli cells were built, expressed and purified. The new DAS-LFIA has given 100% sensitivity and specificity in contrast with ELISA test (the concordance between the new DAS-LFIA strip and ELISA methods was 94.33%) in a trial of 300 clinical serum to detect HCV (Xiang et al.,2012).

Though, for the detection of Theileria annulata infection, a number of DNA-based serological tests already developed like indirect ELISA and competitive ELISA, polymerase chain reaction, reverse line blot and loop-mediated isothermal amplification respectively. Furthermore no one can give results within 10 minutes like the newly developed Ta-LFD(T. annulata lateral flow device) that is very easy to operate as well as user friendly to detect T. annulata infection in the field sample (Abdo et al., 2010).

Ever developed LFIA strips have a diversified application. As a food safety screening tool, a test strip developed to detect 'okadaic acid' in shellfish employing a colloidal gold-monoclonal antibody (McAb) conjugate which gives the result within 10 minutes (Lu et al., 2012). Similarly, another strip developed in competitive format that can detect olaquindox residues in the urine and muscle tissues of pig. The timing of the test is only 5-8 minutes (Song et al, 2011). Cyromazine and melamine in animal origin foods also could be screened by LFIA test strip (Le et al., 2013). An antibacterial medicine using in poultry named sulphonamides which is very harmful for human body could be detect within 10 minutes from the eggs and muscle of chicken (Wang et al., 2007). In addition, another LFIA test strip developed to ensure the presence of enrofloxacin (ENR) residues in muscles chicken (Zhao et al., 2008). In agriculture, to detect atrazine (pesticide residues), scientists developed a strip which can give results within 5 minutes in agriculture field (Kaur et al., 2007). However, LFIA strip also developed for pollution detection (Liu et al., 2012). LFIA strip can detect potato virus X (PVX) in potato leaf tissue and sprout extracts (Drygin et al., 2012).

The concept of LFIA on a paper strip is going to be shifted by 'thread' in near future. For multiplexed quantitative high-sensitivity diagnosis, a new technology for sandwich LFIA called 'Immunochromatographic Assay on Thread' (ICAT) has been developed (Zhou et al., 2012).

Conclusion

Overall, LFIA is a popular and on-site diagnosis method getting excellence in its procedure day by day. The double sandwich LFIA, the concept of ICTA (Assay on Thread), multiplexing for multi-analyte detection by using quantum dots or up-converting phosphor, may give a new pace in the research field and application of Lateral Flow Immunoassay.