Essays in Biochemistry (2016) 60 111–120

DOI: 10.1042/EBC20150012

Lateral flow assays

Katarzyna M. Koczula and Andrea Gallotta
Xeptagen SpA, VEGA Science Park, Via delle Industrie 9, Venice, Italy
Correspondence: Andrea Gallotta ([email protected])

Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable
detection devices popular in biomedicine, agriculture, food and environmental sciences.
This review presents an overview of the principle of the method and the critical compo-
nents of the assay, focusing on lateral flow immunoassays. This type of assay has recently
attracted considerable interest because of its potential to provide instantaneous diagnosis
directly to patients. The range and interpretation of results and parameters used for evalu-
ation of the assay will also be discussed. The main advantages and disadvantages of LFAs
will be summarized and relevant future improvements to testing devices and strategies will
be proposed. Finally, the major recent advances and future diagnostic applications in the
LFA field will be explored.

Introduction
The lateral flow assay (LFA) is a paper-based platform for the detection and quantification of analytes
in complex mixtures, where the sample is placed on a test device and the results are displayed within
5–30 min. Low development costs and ease of production of LFAs have resulted in the expansion of its
applications to multiple fields in which rapid tests are required. LFA-based tests are widely used in hos-
pitals, physician’s offices and clinical laboratories for the qualitative and quantitative detection of specific
antigens [1] and antibodies [2], as well as products of gene amplification [3,4]. A variety of biological sam-
ples can be tested using LFAs, including urine [5], saliva [6], sweat [7,8], serum [9], plasma [10], whole
blood [10,11] and other fluids. Further industries in which LFA-based tests are employed include vet-
erinary medicine [12], quality control [13], product safety in food production [14], and environmental
health and safety [15]. In these areas of utilization, rapid tests are used to screen for animal diseases [16],
pathogens [17,18], chemicals [19], toxins [20] and water pollutants [21,22], among others.
In recent years there has been an increasing demand for point-of-care multiple diagnostic assays with
multiple test lines allowing the rapid and simultaneous detection of multiple analytes present in samples.
Such assays (potentially a single LFA) should be easy to perform without the use of laboratory investi-
gation, or individuals trained in chemical analysis. LFAs are very good candidates as they are cheap to
produce, easy to use and, importantly, widely accepted by users and regulatory authorities. As the path-
way for the development and introduction of novel technologies to the clinical diagnostics market requires
hundreds of millions of dollars and decades of work, the improvement and further development of already
established LFA technologies is a favourable alternative. This process has the potential to produce devices
that may become powerful tools for new challenging applications such as early cancer detection. More-
over, because of the long shelf life and the fact that refrigeration is not required for their storage, LFA
are very well adapted for use in developing countries, small ambulatory care settings, remote regions and
battlefields.
Depending on the elements of recognition used, LFAs can be categorized into different types (Figure 1).
This review focuses on ‘lateral flow immunoassays’ (LFIAs), in which antibodies are exclusively used as
Version of Record published recognition elements. Nucleic acid LFA are used for the detection of amplicons which can be formed
30 June 2016 during the polymerase chain reaction (PCR) [23].


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 Essays in Biochemistry (2016) 60 111–120
DOI: 10.1042/EBC20150012

Figure 1. Classification of lateral flow assays

Based on the recognition elements involved in the assay, we can distinguish the lateral flow immunoassay (LFIA) and the nucleic acid lateral
flow assay (NALFA).

Principle of the lateral flow immunoassay

The principle behind the LFA is simple: a liquid sample (or its extract) containing the analyte of interest moves with-
out the assistance of external forces (capillary action) through various zones of polymeric strips, on which molecules
that can interact with the analyte are attached. A typical lateral flow test strip (presented in Figure 2) consists of over-
lapping membranes that are mounted on a backing card for better stability and handling. As shown in Figure 3, the
sample is applied at one end of the strip, on the adsorbent sample pad, which is impregnated with buffer salts and
surfactants that make the sample suitable for interaction with the detection system. The sample pad ensures that the
analyte present in the sample will be capable of binding to the capture reagents of conjugates and on the membrane.
The treated sample migrates through the conjugate release pad, which contains antibodies that are specific to the
target analyte and are conjugated to coloured or fluorescent particles – most commonly colloidal gold and latex mi-
crospheres. The sample, together with the conjugated antibody bound to the target analyte, migrates along the strip
into the detection zone. This is a porous membrane (usually composed of nitrocellulose) with specific biological
components (mostly antibodies or antigens) immobilized in lines. Their role is to react with the analyte bound to
the conjugated antibody. Recognition of the sample analyte results in an appropriate response on the test line, while
a response on the control line indicates the proper liquid flow through the strip. The read-out, represented by the
lines appearing with different intensities, can be assessed by eye or using a dedicated reader. In order to test multiple
analytes simultaneously under the same conditions, additional test lines of antibodies specific to different analytes
can be immobilized in an array format [24,25]. On the other hand, multiple test lines loaded with the same antibody
can be used for semi-quantitative assays. The principle of this ‘ladder bars’ assay is based on the stepwise capture of
colorimetric conjugate–antigen complexes by the immobilized antibody on each successive line, where the number
of lines appearing on the strip is directly proportional to the concentration of the analyte [26–28]. The liquid flows
across the device because of the capillary force of the strip material and, to maintain this movement, an absorbent pad
is attached at the end of the strip. The role of the absorbent pad is to wick the excess reagents and prevent backflow
of the liquid.
Two formats of the LFIA can be distinguished: direct and competitive. A direct test is used for
larger analytes such as the p24 antigen used in the human immunodeficiency virus (HIV) test ([30] and
http://www.orasure.com/products-infectious/products-infectious-oraquick.asp), as well as analytes with multiple
antigenic sites such as human chorionic gonadotropin (hCG) used in pregnancy tests [31]. The hCG test is an ex-
ample of a sandwich-based assay, where the target is immobilized between two complementary antibodies. In the
direct test, the presence of the test line indicates a positive result and the control line usually contains species-specific
anti-immunoglobulin antibodies, specific for the antibody in the particular conjugate. In the case of small molecules
with single antigenic determinants, which cannot bind to two antibodies simultaneously, competitive tests are used.
In this type of test, the analyte blocks the binding sites on the antibodies on the test line, preventing their interactions

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Figure 2. Typical configuration of a lateral flow immunoassay test strip

LFA is usually composed of the following elements: sample pad, conjugate release pad, membrane with immobilized antibodies and adsor-
bent pad. The components of the strip are usually fixed to an inert backing material.

Figure 3. Operation of a lateral flow immunoassay

(A) Schematic representation of the assay’s mechanism. Top: the sample is deposited on the sample pad and migrates towards the con-
jugate. Middle: the conjugated antibodies bind the target analyte and (bottom) migrate to the test line, where the bound target analyte is
captured. (B) The most commonly used LFA is the pregnancy test (One Step hCG Urine Test), which uses hCG strips. Possible results and
interpretation of the test are shown. In the case of a weak positive result, it is recommended to repeat the test 1 week later. Ab, antibody.

with the coloured conjugate. Therefore, a positive result is indicated by the lack of signal in the test line, while the
control line should be visible independently of the test result.

Components of the assay

The most common difficulties in the manufacture of LFA devices are caused by the hidden complexity of the device. As
the test is composed of many elements, problems can be caused by material incompatibility, flaws in the connection of
the overlapping elements or imperfect material characteristics. During the development of LFIAs, most attention has
been focused on finding the most suitable detection method or choosing the best antigen or antibody. Nonetheless,
it is very important to pay attention to all elements of the test, including the basic components such as the backing
card, adhesive strip and cover tape, in order to produce a consistent and high-quality product.


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Antibody
Although the physical components of the test strip, construction techniques and buffers play the major role in opti-
mizing the test, the heart of these processes are the antibodies, which need to be carefully designed and highly purified.
It is very important to ensure a consistent antibody supply with proven affinity and specificity. Use of monoclonal
antibodies (mostly derived from mouse hybridomas) is preferable, as it allows the production of specific antibodies
in large quantities.

Label
The most important requirements of the nanoparticle label include:
• colloidal stability in solution under various conditions and temperatures
• susceptibility for detection over a large (and useful) dynamic range
• efficiency and reproducibility of conjugation (without the loss of chemical and biological integrity and activity)
• lack of or very low non-specific binding characteristics (ensuring a high signal-to-noise ratio)
• commercial availability at low cost
• easy and scalable conjugation procedure.
Nowadays colloidal gold is the most widely used label in commercial LFIA. Although it can be prepared in the
laboratory at low cost, there are many commercial sources available. It has an intense colour and no development
process is needed for visualization. Moreover, it has high stability in both liquid and dried forms. Another popular
label is latex, which can be tagged with a variety of detector reagents such as coloured or fluorescent dyes, and magnetic
or paramagnetic components. As latex can be produced in multiple colours, it has an application in multiplex assays,
which require discrimination between numerous lines. Carbon and fluorescent labels, or enzymatic modification
[32,33] of the labels, are also used to improve the sensitivity of the assay. Carbon nanotubes have been shown to
exhibit a limit of detection that is 10-fold lower than that of gold [34]. Fluorescent nanoparticles such as quantum
dots may result in a high background noise which has been shown to be overcome by polymer encapsulation and
surface blocking [35].

Membrane
The membrane is considered the most critical element in LFA strips and nitrocellulose is by far the most commonly
used material. Moreover, there are also ‘pillar-based’ capillary LFA devices used for deoxyribonucleic acid (DNA)
hybridization detection (where micropillar arrays replace the membrane), which have the advantage of more precise
control of the capillary flow [36]. Important parameters characterizing a good membrane material are the capillary
forces, as well as the ease of binding and immobilizing proteins necessary for subsequent selection, reaction and
detection. A range of nitrocellulose pore sizes are available, from 0.05 to 12 μm. However, as the pores are not equally
distributed (because of the manufacturing process), capillary flow time is a more accurate parameter and it should
be used when selecting the most effective strip material. The capillary flow time is the time required for the liquid to
travel to and completely fill the strip of the membrane.

Sample pad
The sample pad can have multiple roles, the most important of which is to evenly distribute the sample and to direct it
to the conjugate pad. The sample pad is usually impregnated with buffer salts, proteins, surfactants and other liquids
to control the flow rate of the sample and to make it suitable for the interaction with the detection system. Moreover,
the pores of the sample pad can act as a filter in order to remove redundant materials, e.g. red blood cells (Figure 4).

Conjugate pad
The main role of the conjugate pad is to hold the detector particles and keep them functionally stable until the test is
performed. This is ensured by the composition of the conjugate buffer, containing carbohydrates (such as sucrose),
which serve as a preservative and a resolubilization agent. When the conjugate particles are dried in the presence of
sugar, the sugar molecules form a layer around them stabilizing their biological structures [37]. When the sample
enters the conjugate pad, the sugar molecules rapidly dissolve carrying the particles into the fluid stream. It is crucial
that the release is consistent between individual test strips.

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Figure 4. An example of an integrated sample collection device combined with a cassette and strip reader
The device is designed for easy collection of a whole blood sample and running of a quantitative test strip. Red blood cells are mechanically
separated on the sample pad. (Pictures adapted from http://www.dcndx.com and https://www.youtube.com/watch?v=FvIIozN58gw).

Absorbent pad
The role of the absorbent pad is to wick the fluid through the membrane and to collect the processed liquid. The
absorbent pad allows the use of larger sample volumes, which results in increased test sensitivity. The most popular
absorbent pads are made of cellulose filters.

Detection methods
Since the LFIA is an antibody-based technique, specificity and sensitivity may be affected by other chemicals with
similar structures, leading to false positive results. The sensitivity of assays is limited by the K d (dissociation constant)
of the antibody–antigen conjugate and by the colorimetric read-out. In order to overcome these limitations, both read-
ers and novel biochemical techniques have been developed to improve product quality and customer convenience.
The selection of a detection system is mainly determined by the label employed in the analysis. Fluorescent dyes or
paramagnetic particles cannot be detected directly by the naked eye and require dedicated readers for quantitative
analysis (Table 1). Moreover, automated detection methods provide advantages over manual imaging and processing
in terms of time consumption, interpretation of results and adjustment of variables.

Advantages and disadvantages of LFAs

Many LFIAs are designed for use at point-of-care/need, providing cheap, rapid and easy tests desirable in many in-
dustries. However, regulatory bodies often require confirmation of results using an independent method. Therefore,
LFIA are only suitable for primary screening at point-of-care/need. Because of their long shelf life and the fact that
refrigeration is not required for storage, these tests are very well adapted for use in developing countries. As the visual
result is usually clear and easily distinguished, no additional specific equipment is needed. A summary of the advan-
tages and disadvantages of LFAs is presented in Figure 5. Research is on-going to address some of the key weaknesses


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DOI: 10.1042/EBC20150012

Table 1. The most commonly used detection methods employed in lateral flow assays
Examples of applications of these systems can be found in optical readers [45,46], camera readers [47], ladder bars [26],
fluorescent readers [48], chemiluminescent readers [49] and electrochemical readers [50]. Examples of labels include:
fluorescent [51,52], paramagnetic [47,53], enzyme [54,55] and carbon nanoparticles [34].

of LFAs, especially with respect to quantitative results. Data can be digitized using scanners or cameras with dedi-
cated software, which will also allow the documentation of results. However, technological improvements will affect
the cost of apparatus and the duration of analysis.

New strategies in LFAs

In recent years, the major advances in LFA development have included novel signal-amplification strategies, applica-
tions of new labels, improved quantification systems and simultaneous detection. Some of the new strategies used to
enhance the signal from the colloidal gold nanoparticles (GNPs) have adopted silver enhancement technology [38,39]
or combinations of GNPs with an enzyme (such as horseradish peroxidase), which results in catalytic amplification of
the signal [33]. To improve the detection sensitivity, novel reagents have been identified, including magnetic particles
such as nano-gold microspheres, or immune-nanoparticles, which reduce the detection limits to at least 0.1 ng/ml
[40]. Another way to increase assay sensitivity is the implementation of a suitable quantity system such as a thermal
contrast, laser or light-emitting diode (LED), which can result in signal amplification up to 1000-fold [41]. Some
successful developments of simultaneous detection techniques have been described. These include a combination of
colloidal gold nanoparticles and oligonucleotides for the simultaneous detection of antigens and antibodies [42] and
the use of two conjugate pads for the simultaneous detection of two proteins [43]. Moreover, combinations of LFAs
with computational methods have led to the first example of combinations with electronic logic gates such as ‘OR’
and ‘AND’, providing a novel logic-sensing platform [44].

Discussion
In order to address the criteria demanded by next-generation diagnostic markets, some of the fundamental fea-
tures of LFAs must be improved. First, assays need to be more reproducible and sensitive, easier to manufacture
and operate, and most importantly from a clinical point of view, they should provide relevant results that correlate
with other laboratory-based diagnostic systems. Automation of the manufacturing process and sample application,
as well as improved read-out and data processing, are required to achieve these aims. Moreover, material science
should be applied to bring novel more appropriate custom-designed materials into use, as well as the introduction of
new labelling and reading technologies. The use of new labels such as quantum dots and the upconverting phos-
phors (microscopic ceramic powders that convert infrared light wavelengths into visible coloured light) will im-
prove sensitivity, allowing the use of samples with lower concentrations of the analyte such as sweat or salvia. In

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Figure 5. Advantages and disadvantages of the lateral flow assay

Adapted from [49].

the Western world, integration of LFA into a lab-on-a-chip design may bring additional advantages, but will also in-
crease costs. For the non-laboratory-based applications, the LFAs should remain simple and affordable; however,
good recognition elements must be available and visual qualitative (on/off) or semi-quantitative results must be
sufficient.

Conclusion
The unique and remarkable properties of LFAs have contributed to the detection of disease biomarkers and infectious
agents in medicine, agriculture, food and environmental safety. Although the principle of the method has remained
unchanged for decades, there have been continuous improvements of LFA techniques leading to increased sensi-
tivity and reproducibility, and the simultaneous detection of several analytes. Importantly, these assays can now be
effectively performed outside the laboratory, providing great advantages for use in developing countries and at the
point-of-care, whether in the field or in more traditional clinical settings.


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Summary
• Lateral flow assays (LFAs) can be used for the detection of proteins, haptens, nucleic acids and amplicons.
• LFAs are well established as a valuable tool in medical, veterinary, food, agricultural and environmental
settings and for use in industrial diagnostics.
• The principle of an LFA is based on the movement of a liquid sample though a polymeric strip with attached
molecules that interact with the analyte, providing a signal that can be visually detected.
• Although the concept behind the LFA is simple, the device has a complex architecture and many critical
elements need to be considered during instrumental design.
• The most critical elements of the assay are the antibodies and the membrane, but attention should be paid
to all of the materials used to ensure the compatibility and consistency of the product.
• An LFA is a fast, low cost, portable and easy-to-use assay; however, the results are mostly qualitative (on/off)
or semi-quantitative.
• An LFA is usually used for initial screening, which can be confirmed later by a fully quantitative method.
• LFA devices can be evaluated using parameters such as sensitivity, specificity and efficiency.
• Recent advances and future goals for improving LFAs are focused on identifying new signal amplification
strategies, nanoparticle labels and quantification systems, as well as improving simultaneous detection.

Abbreviations
GNP, gold nanoparticle; hCG, human chorionic gonadotropin; LFA, lateral flow assay; LFIA, lateral flow immunoassay.

Funding
We acknowledge funding from the European Commission Framework Programme 7 through the Marie Curie Initial Training Net-
work PROSENSE [grant number 317420, 2012–2016].

Competing Interests
The Authors declare that there are no competing interests associated with the manuscript.

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c 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.