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The assessment aims to give a brief account on the basic principle of Lateral Flow Immunoassay, its types and diversified applications that acquired extensive research concentration and consumer interest. The ideas of diagnostic tests using body fluids were practiced long back and the notion of researches on rapid immunochromatographic tests were found back to the 1950s (O'Farrell, 2009 cited in Wong and Tse, 2009). From the mid-1960s, immunoassays have been started to be used in diagnostics, industry, laboratory and its development research was carried on simultaneously (Rosen, 2009 cited in Wong and Tse, 2009). Immunoassays depend on the interaction between antigen and antibody, their binding and production of measurable signal. Among the different types of immunoassays, lateral flow immunoassay (LFA) is one of the gifts of invention that made modern diagnostic tools enriched and fast. It is simple but specific and reliable method of diagnosis where the biological samples are directly diagnosed using signals resulting from antigen-antibody interaction.
Starting its journey with the procedural similarities to latex agglutination test (Plotz and Singer, 1956), development and evaluation of LFA passed a long way to be established as an actual technology in 1980 and research continued on its conformational and materialistic improvement to make it more rapid, specific and sensitive tool. In the early period, plate-based immunoassays were performed and then came enzyme-immunoassays that were preferable and better option than previously used radioimmunoassays (O'Farrell, 2009 cited in Wong and Tse, 2009).
Human pregnancy test to detect human chorionic gonadotropin (hCG) hormone by radioimmunoassay invented by Paul and Odell in 1964 (Odell et al., 1967) was probably the most exploited test which drew a lot of attention. Extensive research was performed to generate improved antibodies, to understand the actual biology of hCG and eventually, to develop easier and effective LFA strip (O'Farrell, 2009 cited in Wong and Tse, 2009; Posthuma-Trumpie et al., 2009).
A typical LFA strip (produced from nitrocellulose, nylon, polyethersulfone, polyethylene or fused silica) consists of several zones and supported by a platform called backing or backing card. Backing made up of a semi-rigid plastic, a pressure-sensitive adhesive, and a release liner that acts as platform of the exact components of the test. At proximal end of the strip, there provided sample application pad deriving from cellulose or cross-linked silica that receives and treats the sample. Next is the conjugate release pad, also, made of cross-linked silica, bears immobilized particulate conjugate or labelled analyte (antigen or antibody depending on test design). Labelling uses coloured or fluorescent nanoparticles (Rundström et al., 2007) and more recently, quantum dots (Li et al., 2012) or up-converting phosphor technology (Niedbala et al., 2001), all intended to enhance sensitivity without hindering the flow of the fluid. Sample remobilizes and interacts with the conjugate and both migrate to the reaction matrix containing specific regions of test line and control line. Biological components (either antigen or antibody) immobilized in these lines and serve to capture the analyte and the conjugate as these flows to the capture zone due to capillary action of the membrane strip. Control line reacts and proves the validity of the test and the absorbent pad wicks the additional reagents. Results obtained by observing the presence or absence of lines in test zone (O'Farrell, 2009 cited in Wong and Tse, 2009; Posthuma-Trumpie et al., 2009).
Figure-1: Typical configuration of a lateral flow immunoassay test strip. Figure copied and pasted from http://www.biodot.com/images/typical_lateral_flow_assay_.gif.
Lateral flow immunoassay format can be either sandwich (direct) or competitive (inhibition) and each one might be qualitative, semi-qualitative and sometimes quantitative. Competitive LFAs are better suited for detecting small molecules with single antigenic determinant and absence of test line interprets positive result. Sandwich LFA, in contrast, detects larger analytes with multiple antigenic sites and here, absence of test line gives negative result (O'Farrell, 2009 cited in Wong and Tse, 2009; Posthuma-Trumpie et al., 2009).
LFA can be used in detection of contamination in food and feed (Zheng et al, 2012; Le et al., 2013; Laura et al., 2011; Blazkova et al., 2009). For fast screening of aflatoxin B2 (AFB2) in food samples, a qualitative LFA using nano-Fe2O3 particles shielded with gold nanoparticles as detector and activated with monoclonal anti-AFB2 antibodies was introduced (Tang et al., 2009). They mentioned that the competitive assay did not provide any false negative results and it might be a versatile method for detection of food toxins if the target antibody can be controlled.
A quantitative immunochromatographic assay was developed to identify the presence of ochratoxin A (OTA) in some cereals where gold nanoparticles were used to label the conjugate and they found good quantitative determination of OTA in the pretreated samples (Laura et al., 2011). Similarly, another competitive LFA was reported to measure the amount of sulphathiazole residues in honey samples that found highly specific test with no cross-reactivity to other chemically similar antibiotics and might be considered as a qualitative method for the determination of sulphathiazole residues (Guillén et al. 2012). Likewise, a comprehensive LFA was developed for concurrent recognition of Listeria contaminations in food samples (Blazkova et al., 2009). For the evaluation of Listeria monocytogenes and Listeria spp, DNA isolation and PCR were prerequisite and the PCR solutions were directly employed to the one-step assay device. They suggested that the assay could be utilized for the revealing of Listeria spp in various food samples. Furthermore, a newly developed Lateral flow immunoassay (LFA) for quantification of zearalenone (ZEN) in food and feed stuffs using highly sensitive anti-ZEN monoclonal antibody was found to be rapid and sensitive method in comparison with previously reported LFA and can meet the current regulations of ZEN monitoring worldwide (Liu et al., 2012). Besides, another simple gel-dependent immune-afinity assay was demonstrated to recognise chloramphenicol residues in food samples (Yuan et al., 2012).
LFA extended its applicability in detection of viruses, for instance, bovine rotavirus in fecal samples (Al-Yousif et al., 2002), influenza virus (Cazacu et al., 2003; Li et al., 2012), FMDV (Jiang et al., 2010) and so on. Cyprinid herpes virus-3 (CyHV-3) was detected by loop mediated isothermal amplification (LAMP)- nucleic acid lateral flow assay depending on hybridization technology and found it rapid but equally sensitive to agarose gel electrophoresis (Soliman and Mansour, 2010). At the same period, Equine infectious anemia virus (EIVA) was identified using simple immunochromatographic lateral flow (ICLF) test and the results were compared with a commercially available agar gel immunodiffusion (AGID) test (Alvarez et al., 2010). They claimed the LF test precise and rapid and of somewhat similar sensitivity with AGID. In contrast, it was executed in correlation with ELISA to measure bone turnover markers and reported as a relevant method to measure the urinary N-telopeptides and serum C-telopeptides (Kyoung et al. 2013). Similar results were observed for mycotoxin detection in comparison to subsequent plating and PCR analysis (Braun-Kiewnick et al. 2011).
Besides, for the detection of Deoxynivalenol (DON) mycotoxin produced by some species of Fusarium in contaminated cereals, a typical quantitative LFA test kit was developed which requires only about 10 min from sample preparation to result read-out (Liu et al., 2012). Furthermore, a LFA was outlined that is specific for Erwinia amylovora responsible for invasive Fire blight disease of pome fruit (Braun-Kiewnick et al. 2011). The diagnosis took from 2days for laboratory submitted samples to 15min with the immunoassay and had considerable sensitivity to be an utilisable confirmatory test for enhanced phytosanitary control of the disease.
Colloidal gold particles in LFA were reported to be used for the identification and diagnosis of foot-and-mouth disease virus (FMDV) serotypes O, A and Asia 1 (Jiang et al., 2010) and also, in competitive format, for simultaneous detection of cyromazine (CA) and Melamine (MA) in foods of animal origin (Le et al., 2013). Furthermore, gold nanoparticles (AuNPs) as labeling carriers with the horseradish peroxidase (HRP) enzyme was found to enhance the performance of the optical LFA (Zhang et al., 2006). On the other hand, 100-fold improved detection limit was stated in LF-DNA tests when UPT reporter was used instead of colloidal gold (Sabina et al., 2001). However, Qualitative gold particle LFAs can provide quantitative results if giant magnetoresistive (GMR) sensors are used in electronic detection systems (Taton et al., 2009).
Europium (III) chelate microparticles with time-resolved fluorescence were applied to estimate the number of eosinophils and neutrophils in blood sample (Rundström et al., 2007). Moreover, europium chelate-loaded silica nanoparticle labels can improve the potential of LFA to be applicable for sensitive and quantitative immunoassays (Xia et al., 2009). Furthermore, potential of fluorescent europium (III) chelate dyed polystyrene nanoparticles gave enhanced analytical sensitivity than colloidal gold particles in of LFA (Juntunen et al., 2012). Again, superparamagnetic nanoparticle (SPMNP) probe by coupling monoclonal antibodies against fish major allergen parvalbumin (PA) was prepared to develop a quantitative LFA (Zheng et al, 2012). The detection duration was fifteen-times less than typical Western Blot assay and thus concluded to be significantly important for large-scale screening and point-of-care detections. Furthermore, LFA using acid precipitated iron oxide nanoparticles(IONPs) coated with biocompatible polymer to measure the conjugation capacity of IONPs to mouse anti-human IgG(4) antibody (MaHIgG4) was developed. The conjugate found efficient enough to detect brugian filariasis within 15 min of assay time (Nor et al., 2012).
The progress of LFA (both competitive and sandwich) with Up-Converting Phosphor Technology (UPT) labels created new applications of it in point-of-care testing than other labels (Niedbala et al., 2001). It was explored in quantitative measurement of the hepatitis B surface antibody and found to be a responsive, fast and potential method of detecting HBsAb (Li et al., 2008). UPT LF-DNA tests were also found sensitive and competitive alternative of gel electrophoresis and southern blotting to detect the presence of HPV16 in DNA extracts of cervical carcinomas (Sabina et al., 2001; Corstjens et al., 2001). In addition, detection of Yersinia pestis was also reported to be rapid and specific using UPT-based LFA (Yan et al., 2006).
A fast and ultrasensitive integrated lateral flow test strip technique was developed to detect Avian Influenza Virus (AIV) (Li et al., 2012). The sandwich fluorescence immunoassay used label-free, high luminescent quantum dots (QDs) as signal output and gold nanoparticle (NP) labels to be captured in the test zone. This method found to be rapid, sensitive and specific for quantitative detection of AIV in field or point-of-care diagnosis in comparison with gold method of virus isolation. Similarly, for quantitative detection of Alpha fetoprotein (AFP) for diagnosis of primary hepatic carcinoma, a portable fluorescent biosensor integrating quantum dot (QD)-based sandwich type immunochromatography test strip was formulated. Though they found some inconsistent results, yet, they suggested this test promising for diagnosis of AFP (Yang et al., 2011).
Therefore, it can be concluded that the simplicity and speed of lateral flow immunoassay made it an exclusive tool to be diversely used in medical diagnostics, food quality monitoring, contamination or infection detection, identification of noxious components in food or feed, environmental pollution and drug abuse (Posthuma-Trumpie et al., 2009; Wong and Tse, 2009). Continued research on its different components, constituents and technology will make it faster, more specific, sensitive and easy-to-use test for humankind.