Sebastian2016 Immunochromatography Combines

Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: 2231-6876
IMMUNOCHROMATOGRAPHY: FORMATS AND APPLICATIONS
Cherian Sebastian 1*, Helen William2

1
Scholar, College of Pharmaceutical Sciences, Govt. Medical College, Kottayam, Kerala, India
2
Assistant Professor, College of Pharmaceutical Sciences, Govt. Medical College, Kottayam, Kerala, India
ARTICLE INFO ABSTRACT
Article history Immunochromatography is a combination of chromatography and immunoassay. In this
Received 13/07/2016 technique, the antigen-antibody reaction which occurs on a membrane is used to determine
Available online the target analyte in the sample. For this the specific biomolecule against antigen or antibody
12/08/2016 of interest is impregnated in a membrane usually made of nitrocellulose along with some
dyes which produces respective coloured lines according to the presence or absence of target
Keywords analyte. The method has widespread application in detecting large kind of molecules
Immunochromatography, including poisonous substances, pathogens in their smallest levels, hormones, etc. By the
Advantages and application of this principle, presence of disease causing organism in the body can be
Disadvantages, detected in early stages at a relatively lesser cost. This review article provide an overview on
Working, principle, working, formats, advantages, disadvantages and some of the important
Formats, applications of immunochromatography.
Applications.
Corresponding author
Cherian Sebastian
Scholar,
College of Pharmaceutical Sciences,
Govt. Medical College,
Kottayam, Kerala, India
cherian3333@gmail.com
+917736596951
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Please cite this article in press as Cherian Sebastian et al. Immunochromatography: Formats and Applications .Indo American
Journal of Pharmaceutical Research.2016:6(07).
Copy right © 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
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Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Vol 6, Issue 07, 2016. Cherian Sebastian et al. ISSN NO: 2231-6876
INTRODUCTION
Chromatography
Chromatography was invented by the Russian botanist Mikhail Tswett in 1903.He employed the technique to separate various
plant pigments (such as chlorophylls and xanthophylls) by passing solutions of these substances through a glass column packed with
finely powdered calcium carbonate. The separated species appeared as coloured bands; the various pigments migrating through the
column at different rates. The various components (solutes) were isolated by cutting and sectioning of the chalk packing. Tswett
choose to designate the name of such a process of separation as chromatography (chroma-colour, graphein-writing). In modern times a
very large number of separations are being done by this prime technique very effectively, involving most often colourless substance as
well, nevertheless, the original term “chromatography” to designate all such separating procedures has been retained.
At present chromatography represents a diverse group of technique which allow the separation of closely related components of the
complex mixtures, hither to inseparable by any other means. In all such techniques, the sample is moved in a mobile phase, may be a
gas, a liquid or, a supercritical fluid. Such a mobile phase is then allowed to flow through an immiscible stationary phase, that is fixed
in place in a column, or, on a planar solid surface. It is defined as: “chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one of which is stationary (stationary phase), while the other, the
mobile phase moves in definite direction" Chromatographic technique have revolutionized the capabilities and extended the frontiers
of analytical chemistry very widely.
Mechanisms of Chromatography
Adsorption Chromatography
The stationary phase is a solid on which the sample components (or solute) are absorbed. The mobile phase may be a liquid
(liquid-solid chromatography) or a gas (gas-solid chromatography). The components distribute themselves between the two phases
through the combination of sorption and desorption processes.
Partition Chromatography
It is most widely used mechanism in gas chromatography; the partitioning of the solute occurs between the mobile gas phase
and a stationary liquid phase, either supported on the small particles packed in a column, or bonded on to the inner walls of a capillary
column.
Ion Exchange Chromatography

Ion-exchange chromatography can only takes place in the liquid phase. Here stationary phase is an ion exchange resin; these
are organic polymers with three dimensional network and ion exchange properties are imparted by attaching ionic groups to the
network. The process of ion exchange takes place in the surface of a solid when brought in contact with the solution.
Molecular Exclusion Chromatography

Separation by this method is based upon exploitation of the size (molecular geometry) of the components.
Affinity Chromatography
It is a method of separating biochemical mixtures based on a highly specific interaction such as that between antigen and
antibody, enzyme and substrate, or receptor and ligand. This is the most selective type of chromatography employed [1]. It utilizes the
specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For
example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are
passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later
extracted by changing the ionic strength or P H. The stationary phase is typically a gel matrix, often of agarose; a linear sugar molecule
derived from algae. Usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell lysate,
growth medium or blood serum. The molecule of interest will have a well-known and defined property, and can be exploited during
the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a
solid or stationary phase or medium. The other molecules in the mobile phase will not become trapped as they do not possess this
property. The stationary phase can then be removed from the mixture, washed and the target molecule released from the entrapment in
a process known as elution. Possibly the most common use of affinity chromatography is for the purification of recombinant proteins
[2].
Immunoassays
Immunoassays are bio-analytical methods in which the quantitation of the analyte depends on the reaction of an antigen
(analyte) and an antibody. Principally, these methods are based on a competitive binding reaction between a fixed amount of labelled
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form of an analyte and a variable amount of unlabelled sample analyte for a limited amount of binding sites on a highly specific anti-
analyte antibody. When these immunoanalytical reagents are mixed and incubated, the analyte is bound to the antibody forming an
immune complex. This complex is separated from the unbound reagent fraction by physical or chemical separation technique.
Analysis is achieved by measuring the label activity (e.g. radiation, fluorescence, or enzyme) in either of the bound or free fraction [3].
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Immunoassay methods have been widely used in many important areas of pharmaceutical analysis such as diagnosis of
diseases, therapeutic drug monitoring, clinical pharmacokinetic and bioequivalence studies in drug discovery and pharmaceutical
industries [4].The analysis in these areas usually involves measurement of very low concentrations of low molecular weight drugs,
macromolecular biomolecules of pharmaceutical interest, metabolites , and/or biomarkers which indicate disease diagnosis or
prognosis [5]. The importance and widespread of immunoassay methods in pharmaceutical analysis are attributed to their inherent
specificity, high throughput, and high sensitivity for the analysis of wide range of analytes in biological samples. The detection system
in immunoassays depends on readily detectable labels (e.g. radioisotopes or enzymes) coupled to one of the immunoanalytical
reagents (i.e. analyte or antibody). The use of these labels in immunoassays results in assay methods with extremely high sensitivity
and low limits of detection [6].
Types of Immunoassay
Radioimmunoassay
It is a competitive binding assay in which fixed amount of antibody and radiolabelled antigen reacts in the presence of
unlabelled antigen. The labelled and unlabelled antigens compete for the limited binding sites on the antibody. This competition is
determined by the level of the unlabelled (test) antigen present in the reacting system. After the reaction, the antigen is separated into
‘free’ and ‘bound’ fractions and their radioactive counts measured. The concentration of test antigen can be calculated from the ratio
of the bound and total antigen labels using a standard dose response curve.
Enzyme Immunoassay
Enzyme immunoassay (EIA) is analogous to RIA except that the label is an enzyme rather than a radioisotope. The basic
approach for use of an enzyme as an immunoassay label is appreciated by coupling an enzyme molecule into one of the
immunoanalytical reagents (analyte or antibody), by appropriate chemical technique, and then carrying out the immunoanalytical
reaction in the normal way. Following the separation of bound and free fractions, the enzyme activity is monitored in either of the two
fractions. This is achieved by adding substrate, and subsequent monitoring the turnover of the substrate to product.
Fluoroimmunoassay
Fluoroimmunoassay (FIA) is analogous to RIA except that the label is a fluorophore rather than a radioisotope.
Chemiluminescence Immunoassay
Chemiluminescence immunoassay (CLIA) involves a chemiluminescent substance as a label. The growing success of this
technique in pharmaceutical analysis due to its high performance, low detection limits, and good precision.
Liposome Immunoassay
Liposome immunoassay (LIA) is the assays involving a liposome-encapsulating marker. In LIA, liposomes are prepared and
then coupled to either analyte or antibody by a suitable procedure, and then carrying out the assay in normal way. Detection in LIA
relies on the lysis of the liposome and releasing the encapsulated marker, which is then measured and related to the analyte
concentration.
Immunoelectroblot technique
Immunoelectroblot or electroimmunoblot techniques combine the sensitivity of enzyme immunoassay with much greater
specificity. The technique is a combination of three separate procedures: (a)separation of ligand-antigen components by
electrophoresis, (b).blotting of electrophoresed ligand fraction on nitrocellulose membrane strips; and (c).enzyme immunoassay to
detect antibody in test sera against various ligand fraction bands or probe with known antisera against antigen bands
Immunochromatographic Tests
The test system is a small cassette containing a membrane impregnated with anti-antibody-colloidal gold dye conjugate. The
membrane is exposed at three windows on the cassette. The test serum is dropped at the first window. As serum travels upstream by
capillary action, due to formation of antibody-conjugate complex, a coloured band appears indicating positive test [7].
IMMUNOCHROMATOGRAPHY ASSAY PRINCIPLE

Immunochromatography is one of the most important and effective technique in the detection of virus and bacteria; It plays an
important role in the diagnosis. These assays are also known as lateral flow test or simply strip test which are the devices intended to
detect the target analyte in sample without the need for specialized and costly equipment. They are the logical extension of the
technology used in latex agglutination tests, the 1st of which was developed in 1956 by Singer and Plotz. The principle is based on
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dye labelled antibody specific for target analyte which is present on the lower end of nitrocellulose strip or in the plastic well along
with the strip. The antibody which is specific for target antigen is also bound to the strip in a thin test line and antibody antigen
specific for labelled antibody bound to control line. So when the sample and buffer are placed on strip or in a well-mixed with labelled
antibody to draw across the lines of bound antibody.
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This gives the identification of antigen present or absent. If the antigen is present, then some of the labelled antibody will be
trapped on the test line and the excess labelled antibodies are trapped on the control line. Currently, the principles governing this test
are being extended and forwarded to allow for some exciting new possibilities for future test [8]. Undoubtedly, (immuno)affinity
chromatography is one of the most powerful techniques to selectively isolate and or concentrate minor components of interest from a
complex mixture.Its selectivity is derived from the use of an immobilised specific biomolecule, the affinity ligand, on a suitable solid
phase support. Ideally, a sample passed through an (immuno) affinity column, separates into two bands. The first band elutes with a
capacity ratio k’=0 and contains all the compounds which do not bind to the affinity ligand. The second band, containing only the
analyte, should be strongly adsorbed to the ligand and should not elute. Increasing the mobile phase strength by a change of pH or
other parameter causes the analyte peak to elute. If used in combination with other modes of chromatography, for instance high
performance liquid chromatography (HPLC), the immunoaffinity column can serve as selective on-line pre-cleanup step for the
isolation of a group of compounds which are captured by one or more immobilised antibodies. After elution of the captured analytes,
quantitative analysis can be performed by HPLC. Via this way the advantages of both techniques, high selectivity as well as high
precision, are combined [9]. The benefits of immunochromatographic tests include:
1. User-friendly format.
2. Very short time to get test result.
3. Long-term stability over a wide range of climates.
4. Relatively inexpensive to make.
These features make strip tests ideal for applications, such as home testing, rapid point of care testing, and testing in the field
for various environmental and agricultural analytes. In addition, they provide reliable testing that might not otherwise be available to
developing countries. Basically, any ligand that can be bound to a visually detectable solid support, such as dyed microspheres, can be
tested for qualitatively, and in many cases, even semi-quantitatively. Some of the more common lateral flow tests currently on the
market are tests for pregnancy, Strep throat, and Chlamydia. These are examples of conditions for which a quantitative assay is not
necessary [10].
LATERAL FLOW ASSAY VERSUS IMMUNOCHROMATOGRAPHY

The concept of immunochromatography as a combination of chromatography (separation of components of a sample based
on differences in their movement through a sorbent) and immunochemical reactions emerged a long time ago, and it has been
implemented in many different ways. Today, the most widespread immunochromatographic system is the test strip – an assembly of
several plain porous carriers impregnated with immunoreagents. On contact with the test strip, a liquid sample flows laterally along
the carriers, and detectable immune complexes are formed in certain zones of the test strip. Test strips are widely used for the early
detection of pregnancy, for drug screening, to identify markers for various diseases, and for a number of other analytical tasks.
However, a few decades ago, the term ‘‘immunochromatography’’ was used to describe a different type of analysis (i.e., separation of
samples on a column containing a sorbent with covalently-bound antibodies specific to a target substance). This approach is used in
modern analytical practice to separate and to concentrate various substances, these two types of immunoassay systems are very
different. However, it is difficult to consider them as fundamentally different methods when they share the same analytical
components and the same name. In the broad sense of the term, ‘immunochromatography’ analytical systems combine longitudinal or
transverse liquid flow through a carrier with immunochemical reactions. What distinguishes immunochromatography assays from
other types of immunoassays? Regardless of the format, all immunochromatography systems consist of immuno reagents immobilized
on a carrier and fluid flow through that carrier.
This approach allows for:

1. Adjustable and rapid formation of immune complexes;
2. Removal of unreacted compounds from the binding zone during the analysis; and
3. The use of special zones to concentrate and to detect target complexes.
Immunochromatography combines advantages of homogeneous and heterogeneous analytical methods. It combines the speed
of a homogeneous immunoassay with the separation of reacted and unreacted compounds by a variety of heterogeneous methods.
Another advantage of immunochromatography is that the fluid flow through the carrier (e.g., sorbent and membrane,) enables
separation of reacted from unreacted products without the need for additional precipitation or washing steps. Immunochromatographic
analyses are rapid and simple, allowing for point-of-care testing. These advantages explains their success in medical diagnosis.
Immunochromatographic test strips are mass produced, and are widely used to detect certain cancer and cardiac markers and
infectious microorganisms. They are also used in serodiagnostic analyses to identify antibodies against various pathogens. There are
many companies manufacturing a variety of immunoassay products for medical and veterinary diagnostics.
The market for medical test systems doubled during the period 2003–09, and, despite a slight slow-down caused by macroeconomic
factors, it continues to grow at an average rate of 6% per year. The availability of established technologies to synthesize reagents and
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the relatively inexpensive equipment required to produce test strips (dispensers, cutters, laminators) mean that it is relatively easy to
produce immunochromatographic kits for other applications. The development of immunochromatographic systems for food safety
and quality control is a relatively new field of research. There have been several reviews of recent progress in this area. Most of them
described research related to the development of systems to detect a group of food contaminants or a particular compound, and
discussed factors related to increasing the speed and the sensitivity of the proposed assays [11].
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ADVANTAGES
1 Ease of device preparation
2 Low cost
3 Stability over a wide range of environmental conditions and very long shelf life.
4 Simple and user friendly operation
5 Requirement of small sample volume
6 Most of the time, allows sample application without Pre-treatment
7 Versatility of formats, bio-recognition molecules, labels and detection systems.
8 Less time of analysis
9 Comparable or better sensitivity and specificity than other well established methods
10 High potential of commercialization
11 Easy integration with electronics
12 Wide range of applications
13 No or very little energy consumption
DISADVANTAGES
1 Mostly qualitative or semi-quantitative
2 Reproducibility varies from lot to lot
3 Most of the devices can detect more than one or two analytes simultaneously
4 Suffers from low biomolecules affinity toward analytes and tendency of cross-reactivity
5 Sometimes, pre-treatment of sample is required which is time consuming
6 Once sample is applied to the strip, capillary action cannot be decreased or speeded up.
7 Analysis time is also dependent on nature of sample itself i.e. viscosity, surface tension [12].
WORKING OF IMMUNOCHROMATOGRAPHIC ASSAYS

Sample placement
To perform the test the sample is placed on the one the sample pad at one end of the strip. The sample may be used alone as
is commonly done with urine or serum or whole blood or plasma compatible tests, or it may be mixed with a buffer specific to the test.
Solubilisation of detector molecules

With the addition of the sample, the detector molecules (bio-recognition molecule) are solubilised. After which the detector
molecules mix with and bind to the analyte in the sample (if analyte is present).
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Capillary action
The capillary action draws the fluid mixture up the sample pad and into the membrane. The sample or detector molecule
mixture continue to move up the membrane until it reaches the analyte capture molecule. In these lines a second (and third) antibody
or antigen, immobilised as a thin strip in the nitrocellulose will then capture the complex if it is positive for the target analyte. The
control line should always show as a visible line, otherwise the test is invalid and must be repeated. If it is positive, a coloured
(typically pink or purple) line develops along with the control line.
Excess absorbed
Excess buffer along with any reagents not captured at the test or control line will then move into the absorbent wicking pad
[13].
The components of the strip are usually fixed to an inert backing material and may be presented in a simple dipstick format or
within a plastic casing with a sample port and reaction window showing the capture and control zones. The technology is based on a
series of capillary beds, such as pieces of porous paper or sintered polymer. Each of these elements has the capacity to transport fluid
(e.g., urine) spontaneously. The first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the
fluid migrates to the second element (conjugate pad) in which the manufacturer has stored the so-called conjugate, a dried format of
bio-active particles (see below) in a salt-sugar matrix that contains everything to guarantee 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.
While the sample fluid dissolves the salt-sugar matrix, it also dissolves the particles and in one combined transport action the sample
and conjugate mix while flowing through the porous structure. In this way, the analyte binds to the particles while migrating further
through the third capillary bed. This material has one or more areas (often called stripes) where a third molecule has been immobilized
by the manufacturer. By the time the sample-conjugate mix reaches these strips, analyte has been bound on the particle and the third
'capture' molecule binds the complex. After a while, when more and more fluid has passed the stripes, particles accumulate and the
stripe-area changes colour. Typically there are at least two stripes: one (the control) that captures any particle and thereby shows that
reaction conditions and technology worked fine, the second contains a specific capture molecule and only captures those particles onto
which an analyte molecule has been immobilized. After passing these reaction zones the fluid enters the final porous material, the
wick, that simply acts as a waste container [14]. 6405
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COMPONENTS USED
A typical lateral flow rapid test strip consist of the following components:
Sample pad
Purpose
One of the major advantages of the lateral flow concept is that these assays can be run in a single step with many different
sample types in a variety of application areas. Sample types can be as diverse as whole blood from a postpartum mother, a sputum
sample from a potential TB sufferer, or a sample of ground beef from a bulk container. Much of the burden of making those samples
compatible with the rest of the assay system falls on the sample application pad. The role of the sample pad is to accept the sample,
treat it such that it is compatible with the assay, and release the analyte with high efficiency. Sample treatments include the filtering
out of particulates or red blood cells, changing the pH of the sample, actively binding sample components that can interfere with the
assay, and disrupting matrix components, such as mucins, in order to release the analyte to the assay. The material chosen to fulfil any
or all of these functions can have a great effect on assay performance due to the inhomogeneity of many available materials and the
type of binders they contain. The method of pad pre-treatment is typically via immersion and drying as with the conjugate pad and, if
such treatment is required, the method must be carefully designed to avoid introducing sources of variation, including buffer
concentration gradients and edge effects upon drying.
Material:
The materials used for the sample pad depend on the requirements of the application. Examples of such materials are
cellulose, glass fibre, rayon, and other filtration media.
Capacity:
The sample pad material must be treated with assay buffer and other components and dried prior to use. It must also be able
to accept all of the sample volume applied to it in a controlled way, thereby helping to channel fluids into the assay materials rather
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than allowing flooding or surface flow.

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Strength:
The sample pad material should be strong enough to be handled in manufacturing. An important consideration is tensile
strength while wet. If this material is to enter high-volume production, it must endure the tension, without breaking, from a reel-to-reel
production system while being immersed in a tank of fluid. The immersion of the pad occurs as part of the pad pre-treatment, where
the pads are impregnated with an assay buffer containing pH buffer, surfactants, blocking reagents (if required), additives, and other
reagents to increase sensitivity of the assay. In some cases, the sample pad and the conjugate pad can be the same unit, although this is
not common. Typically, the conjugate and the assay buffer are not compatible. However, it is not unusual to see in some assays the
same material being used for the sample pad and the conjugate pad, although the pads are treated individually and subsequently
assembled [15]. It is made of cellulose and/or glass fibre and sample is applied on this pad to start assay. Its function is to transport the
sample to other components of lateral flow test strip (LFTS). Sample pad should be capable of transportation of the sample in a
smooth, continuous and homogenous manner. Sample application pads are sometimes designed to pre-treat the sample before its
transportation. This pre-treatment may include separation of sample components, removal of interferences, adjustment of pH, etc.
[12].
Conjugate or reagent pad

Purpose
The role of the conjugate pad in a lateral flow immunoassay is to accept the conjugate, hold it stable over its entire shelf life,
and release it efficiently and reproducibly when the assay is run. In practice, variations in conjugate deposition, drying, and release
from the membrane constitute major contributions to the coefficient of variation in assay performance. Assay sensitivity can also be
adversely affected by poor conjugate mixing and release from the conjugate pad. Depending on the system, some may favour fast
release while others favour slow release of the conjugate. However, the release must always be consistent. Because of the nature of the
materials used, it is often necessary to pre-treat conjugate pads to ensure optimal release and stability. Pre-treatment is performed by
immersion of the pad in aqueous solutions of proteins, surfactants, and polymers, followed by drying. This process, similar to
membrane dipping and drying described above, can be performed either in manual batch mode or in continuous inline mode, the latter
giving the best opportunity for homogeneous processing of entire batches of materials. The addition of conjugates to the treated pad is
a critical step for the final performance of the test. Two methods are typically used. The first is immersion of the treated conjugate pad
into the conjugate suspension. The second is dispensing wit quantitative non-contact dispensers such as the BioDot AirJet Quanti 3000
[15]. It is the place where labelled bio-recognition molecules are dispensed. Material of conjugate pad should immediately release
labelled conjugate upon contact with moving liquid sample. Labelled conjugate should stay stable over entire life span of lateral flow
strip. Any variations in dispensing, drying or release of conjugate can change results of assay significantly. Poor preparation of
labelled conjugate can adversely affect sensitivity of assay. Nature of conjugate pad material has an effect on release of labelled
conjugate and sensitivity of assay [12].
Material:
The materials of choice are glass fibers, polyesters, or rayons.
General characteristics
Flow Characteristics:
For best results, the materials must be hydrophilic and allow rapid flow rates. Most materials used in lateral flow
immunoassay systems are very hydrophobic in nature, and must be treated to make them hydrophilic. This is done during the
manufacturing of the assay rather than by the material manufacturer, although there are exceptions to that. This treatment involves the
immersion of the pads in a solution of proteins, polymers, and surfactants, followed by drying at high temperatures.
Release Characteristics:
The conjugate pad must release the conjugates efficiently and reproducibly over the shelf-life of the product. Typically, some variation
in release may occur due to the nature of binding of the particle conjugate to the fibres of the material. It is important during assay
optimization to generate stabilization chemistries that minimize this effect and create the most efficient release of particles possible.
Stability:
The conjugate pad must not destabilize the conjugate over the entire shelf-life (up to 2 years). Typically, some destabilization
does occur, due to the binders present in the majority of these materials. Assay optimization therefore involves the testing of multiple
materials for compatibility with the protein–particle conjugate being used.
Reaction membrane
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Purpose:
The purpose of the analytical region in a lateral flow immunoassay is to bind proteins at the test and control areas and to
maintain their stability and activity over the shelf-life of the product. When the strip is run, it must accept the conjugate and sample
from the conjugate pad, flow them consistently to the reaction area, allow the reaction at the test and control lines to happen, and allow
excess fluids, label, and reactants to exit without binding.
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Material
The material of choice in the vast majority of lateral flow immunoassay systems has historically been nitrocellulose. Several
attempts have been made to introduce other material types into the market, including nylon and polyvinylidene fluoride (PVDF)
membranes. However, those attempts have had limited success, apparently due to factors including cost, limited utility, the need for
education regarding new chemistry and processing requirements, and resistance to change due to the large bank of existing experience
in the use of nitrocellulose. Nitrocellulose, while extremely functional, is not an ideal matrix for an analytical membrane in LFIAs. It
does have certain characteristics that make it useful, and it remains the only material that has been successfully and widely applied in
this way to date. These characteristics include relatively low cost, true capillary flow characteristics, high protein-binding capacity,
relative ease of handling (with direct cast, or backed membranes), and a variety of available products with varying wicking rates and
surfactant contents. However, the material also possesses a variety of characteristics that make it imperfect for this application. These
include imperfect reproducibility of performance within and between lots, shelf-life issues, flammability (primarily in unbacked
membranes), variable characteristics due to environmental conditions, such as relative humidity and being subject to breakage (if
unbacked), compression, and scoring during processing. As a result of these issues with the material, developers and manufacturers
spend a considerable amount of time and effort in optimizing chemistries that overcome some of the inherent material issues and in
developing manufacturing processes that guarantee adequate performance over the entire shelf-life of the product. Careful control of
the key processes of dispensing, dipping, and drying, and attention to chemical and biological treatment of the membrane in order to
prevent the introduction of additional variation into the finished product are critical to success.
General characteristics
Flow Characteristics:
In order to function as the reaction matrix in a lateral flow immunoassay system, the materials must be hydrophilic and have
consistent flow characteristics. Nitrocellulose as a base material is hydrophobic, and is made hydrophilic by the addition of rewetting
agents during the membrane production process. These rewetting agents are surfactants, and the type, amount used, and addition
methods of surfactant differ from manufacturer to manufacturer and also from brand to brand within a manufacturer. These factors can
affect the performance of the assay initially and over time. Not every protein is compatible with every surfactant. This is one reason
for screening multiple membrane types during development. The flow characteristics of nitrocellulose membrane change over time,
primarily due to desiccation of the membranes upon storage. Nitrocellulose membranes can be envisaged as a sponge, with the pores
of the sponge being held open by water. If that water is removed, the pores collapse, disrupting the ability of the membrane to wick
fluids through it. This results in changes and inconsistencies in flow rates over time. As speed directly affects assay sensitivity,
extended run times can produce false positive results. This is a major contribution to the variability in lateral flow immunoassays.
Critical to the proper performance of a lateral flow immunoassay system is the requirement that it binds reactants only at the desired
locations, namely the test and control lines. The protein-binding capacity of a membrane, its interactions with proteins, and the
kinetics of the protein-binding process are the parameters that determine the appropriateness of a given set of proteins for the
membrane and the sensitivity of the resulting diagnostic tests. Proteins bind to nitrocellulose through a combination of electrostatic,
hydrogen, and hydrophobic forces. One of the key elements to the production of sensitive and reproducible assays is the consistent
immobilization of immunologically active proteins to test and control lines. It is known that a majority of the proteins lose much of
their immunological activity after binding passively to the membrane surface, due to their inability to bind covalently or directionally
to nitrocellulose. The commonly accepted model for binding of protein to nitrocellulose is that proteins are initially attracted to the
membrane surface by electrostatic attraction. Long-term bonding is then accomplished by a combination of hydrophobic and hydrogen
bonds. Many factors affect the binding process, and these must be considered when developing assays and processing nitrocellulose
membranes. Some of these factors are listed below:
i Reagent choices
 Non-specific proteins: bulking proteins [e.g., bovine serum albumin (BSA), casein] compete for binding sites.
 Materials that interfere with hydrogen bonding: Formamide and urea interfere with hydrogen bonding.
 Materials that interfere with hydrophobic interactions: Tween and Triton interfere with hydrophobic bonding.
 Polymers such as polyvinyl acetate, polyvinyl pyrrolidone, and poly-ethylene glycol interfere with protein binding by a
combination of these effects.
ii Environment
 Humidity should be optimized for binding (25–50% relative humidity at room temperature).
iii Processing Methods

 Dispensing methods: Contact tip versus non-contact will have effects on how protein binds or spreads through the membrane.
 Drying methods: Forced air oven at elevated temperature versus ambient drying conditions. Drying time and methods can affect
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the rearrangement and activity of proteins on the membrane.
Stability:
The membrane must not destabilize bound proteins at test and control lines for entire shelf-life or change its flow
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Membrane Processing:
Nitrocellulose must undergo several processes before integration into the final device. These include deposition of test and
control line proteins using quantitative dispensers, drying using forced air ovens at elevated temperature, and immersion processes for
blocking. To lay down the test and control line proteins, the membrane is striped with proteins using either contact or non-contact
dispensing systems, and is blocked thereafter to control and stabilize the flow-rates and hydration characteristics and to prevent non-
specific binding. The dispensing method used for the test and control lines must be as quantitative as possible and should not vary
with material hydration or absorption characteristics. Non-contact dispensing methods provide the best solution for quantitatively
dispensing proteins onto nitrocellulose. The purpose of blocking a nitrocellulose membrane is to prevent the binding of proteins and
labelled conjugate to the membrane at areas other than the test and control lines. Blocking also serves other functions, including
maintenance of hydration of membranes, modification of wicking rates, and stabilization of test and control line proteins. Blocking is
typically performed by immersion of the membranes in a solution containing proteins, surfactants, and polymers, and is a relatively
uncontrolled process. The blocking method must be carefully controlled to produce optimal performance in the final product over its
entire shelf-life. Drying is subsequently performed by a combination of blotting to remove surface fluids and with forced air at
elevated temperatures to dry. Again, this drying process must be carefully optimized to minimize variation in the final product.
Availability and Choice:

The correct combination of membrane types and specific proteins is an important factor for the success of a functional test.
Different nitrocellulose membranes can vary considerably in terms of performance characteristics when used with different proteins.
Thus, a variety of suppliers and brands of nitrocellulose membranes are available. Performance of the membrane is typically defined
by factors such as the polymer type used in the membrane, the pore size, the surfactant type, quantity, and the method of surfactant
application. Pore sizes of the membrane used in lateral flow immunoassays range from a nominal 8 to 15 microns, although pore size
is a non-exact descriptor in the case of nitrocellulose membranes. The polymeric structure does not actually create pores, but rather a
tortuous sponge-like pathway for fluid and particle movement. ‘‘Wicking rate’’ or ‘‘capillary rise time’’ is a more appropriate
measure of membrane flow characteristics than pore size. Capillary rise time is defined as the length of time required for a fluid front
to traverse a 40 mm width of membrane and is a manufacturer-defined specification for nitrocellulose membranes. The choice of
wicking rate is important to the kinetics and speed of development of the assay and will have critical effects on assay performance and
sensitivity.
Wick or waste reservoir

Purpose
The wick is the engine of the strip. It is designed to pull all of the fluid added to the strip into this region and to hold it for
the duration of the assay. It should not release this fluid back into the assay or false positives can occur.
Material
The material is typically a high-density cellulose. The choice of wicking material is generally dictated by absorptive capacity,
cost, and caliper. Tensile strength and availability in roll stock should also be considerations [15]. Or otherwise it may be explained as
a further absorbent pad designed to draw the sample across the reaction membrane by capillary action and collect it.
Bio-recognition molecules
Antibodies
Antibodies are employed as bio-recognition molecules on the test and control lines of lateral flow strip and they bind to target
analyte through immunochemical interactions. Resulting assay is known as lateral flow immunochromatographic assay (LFIA).
Antibodies are available against common contaminants but they can also be synthesized against specific target analytes. Mice or other
animals are immunized with target and secreted antibodies are sub-cloned and purified according to application. Antibodies are being
utilized in clinical analysis since five decades for diagnostic needs. An antibody which specifically binds to a certain target analyte is
known as primary antibody but the one which is used to bind a target containing antibody or another antibody is known as secondary
antibody. Process of synthesizing an antibody against toxic analytes is challenging because of toxicity of injected analyte into animal
body which may not be bearable by animal. Antibodies are generally produced from rat or mice and then applied to detect analytes
from human samples. Production and application in different matrix raise serious questions on reliability of analysis. Process of their
generation is strenuous and also temperature sensitive. Affinity of any antibody toward corresponding antibody or another antibody is
known as secondary antibody. Process of synthesizing an antibody against toxic analytes is challenging because of toxicity of injected
analyte into animal body which may not be bearable by animal. Antibodies are generally produced from rat or mice and then applied
to detect analytes from human samples. Production and application in different matrix raise serious questions on reliability of analysis.
Process of their generation is strenuous and also temperature sensitive. Affinity of any antibody toward corresponding antigen is a
concentration dependent factor because of immune response between them and a reasonable response is observed in the range of 10 7
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to 1010 M-1. Concentration of the target analyte is critical in deciding applicability of antibodies as bio-recognition molecules. Limit of
detections as low as nano molar to pico molar range are achievable by using current theoretical methods and changing physical
parameters (amount of reagents on various zones of strip, signal enhancement through modifications on label, pre-incubation of
sample with labeled antibodies) of analysis for a variety of target analytes.
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Aptamers
Aptamers are the artificial nucleic acids and their discovery was reported by two groups in 1990. Aptamers are generated by
an in vitro process known as SELEX (systematic evolution of ligands by exponential enrichment) Aptamers have very high
association constants and can bind selectively with a variety of target analytes Organic molecules having molecular weights in the
range of 100–10,000 Da are outstanding targets for aptamers. Because of their unique affinity toward target molecules, very closely
related interferences can be differentiated. They are preferred over antibodies due to many features which include easy production
process, simple labelling process, amplification after selection, straightforward structure modifications, unmatched stability,
reproducibility and versatility of applications. Their current applications focused on areas of selective chromatography, cell imaging,
target capturing, in vivo therapy, molecular sensing, protein based imaging, cancer cell biology, as enzymes in many biological
applications, cellular physiology, and drug delivery. Specifically in the areas of biosensing, aptamers are used in electrochemical,
florescence, colorimetric, and mass based detection systems.
Molecular beacons
Molecular beacons were first time reported in 1996. Molecular beacons are a special DNA hairpin structure with fluorophore
at one end and quencher at the other end. Fluorophore cannot produce fluorescence in the absence of analyte because of closely
located quencher. But when complimentary DNA sequence is present as a target analyte, stem and loop are opened as a result of a
force and fluorescence signal is observed. Molecular beacons can bind with high specificity and selectivity to nucleic acid sequences,
toxins, proteins and other target molecules. Molecular beacons are composed of 15–30 base pairs in loop which are complimentary to
target analyte and 4–6 base pairs at double stranded stem. Molecular beacons are being used in messenger RNA detection,
intercellular imaging, protein and small molecule analysis, biosensors, biochip development, single nucleotide polymorphism and
gene expression studies
Labels.
List of materials used as a label in LFA is very vast which includes gold nanoparticles, coloured latex beads, magnetic
particles carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, up converting phosphors, organic
fluorophores, textile dyes, enzymes, liposomes and others. Any material that is used as a label should be detectable at very low
concentrations and it should retain its properties upon conjugation with biorecognition molecules. This conjugation is also expected
not to change features of biorecognition probes. Ease in conjugation with biomolecules and stability over longer period of time are
desirable features for a good label. Concentrations of labels down to 10 –9 M are optically detectable. After the completion of assay,
some labels generate direct signal (as colour from gold colloidal) while others require additional steps to produce analytical signal (as
enzymes produce detectable product upon reaction with suitable substrate). Hence the labels which give direct signal are preferable in
LFA because of less time consumption and reduced procedure.
Gold nanoparticles
Colloidal gold nanoparticles are the most commonly used labels in immunochromatography. Colloidal gold is inert and gives
very perfect spherical particles. These particles have very high affinity toward biomolecules and can be easily functionalized. Optical
properties of gold nanoparticles are dependent on size and shape. Size of particles can be tuned by use of suitable chemical additives.
Their unique features include environment friendly preparation, high affinity toward proteins and biomolecules, enhanced stability,
exceptionally higher values for charge transfer and good optical signalling. Optical properties of gold nanoparticle enhance sensitivity
of analysis in lateral flow assay or immunochromatographic assays. Sensitivity is a function of molar absorption coefficient and
accumulation of gold nanoparticles on target molecule. Optical signal of gold nanoparticles in colorimetric LFA can be amplified by
deposition of silver, gold nanoparticles and enzymes.
Magnetic particles and aggregates

Use of magnetic particles as coloured labels in LFA has been reported by number of researchers. Coloured magnetic particles
produce colour at the test line which is measured by an optical strip reader but magnetic signals coming from magnetic particles can
also be used as detection signals and recorded by a magnetic assay reader. It has been reported that magnetic signals are stable for
longer time compared to optical signals and they enhance sensitivity of LFA by 10 to 1000 folds. Fe3O4 particles with small size and
spherical geometry resulted in high sensitivity for detection of Vibrio parahaemolyticus. Major shortcoming of iron oxide
nanoparticles is their drab absorption spectrum which covers whole visible region. Polyethylene glycol modified magnetic iron oxide
particles were changed into different sized aggregates by cross-linking with poly-L-lysine. These aggregates showed better sensitivity
for detection of pesticide paraoxon methyl than individual iron oxide nanoparticles.
Fluorescent and luminescent materials

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Fluorescent molecules are widely used in LFA as labels and the amount of fluorescence is used to quantitate the
concentration of analyte in the sample. Detection of proteins was accomplished by using organic fluorophores such as rhodamine as
labels in LFA. Problem of photo bleaching is linked with organic fluorophores which results in reduced sensitivity. They also suffer
from chemical and metabolic degradation. High photo stability and brightness are required in case of lateral flow assays. Use of
fluorescent europium (III) nanoparticles in LFA showed several folds better sensitivity for detection of free prostate specific-antigen
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than gold nanoparticles.
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Lanthanide chelate-loaded silica nanoparticles were used as a label for detection of Pantoea stewartii subsp. stewartii in
maize and detection limit was 100 folds better than gold colloidal. Other fluorescent labels used in LFA include silica nanoparticles,
and microspheres.
Enzymes
Enzymes are also employed as labels in LFA. But they increase one step in LFA which is application of suitable substrate
after complete assay. This substrate will produce colour at test and control lines as a result of enzymatic reaction. Horseradish
peroxidase labeled antibody conjugates were used for detection of Rabit IgG (R-IgG). Enzymes were also used as labels in LFA to
produce chemiluminescence as a result of reaction with suitable substrate for on field detection of explosives. In case of enzymes,
selection of suitable enzyme substrate combination is one necessary requirement in order to get a coloured product for strip reader or
electro active product for electrochemical detection. In other words, sensitivity of detection is dependent on enzyme substrate
combination. Enhanced LFA sensitivity was observed when enzyme loaded gold nanoparticles were used as a label.
Colloidal carbon
Colloidal carbon is comparatively inexpensive label and its production can be easily scaled up. Because of their black colour,
carbon Nanoparticles can be easily detected with high sensitivity. Colloidal carbon can be functionalized with a large variety of
biomolecules for detection of low and high molecular weight analytes. Colloidal carbon was used as a label in LFA for visual
detection of pesticide methiocarb in surface water. A work was designed to make a comparison between gold nanoparticles, latex
bead, silver enhanced gold, and carbon black nanoparticles as a label for biomolecules for detection of biotin-streptavidin interactions.
Carbon black nanoparticles showed very low detection limits compared to other labels. The sensitivity of LFA employing colloidal
carbon is reported to be comparable with ELISA Presence of irregular shaped large particles and nonspecific adsorption of proteins
and biomolecules are major problems with colloidal carbon [12].
FORMATS
Different formats used in lateral flow assay may be as follows:
Sandwich format
In a typical format, label (Enzymes or nanoparticles or fluorescence dyes) coated antibody or aptamer is immobilized at
conjugate pad. This is a temporary adsorption which can be flushed away by flow of any buffer solution. A primary antibody or
aptamer against target analyte is immobilized over test line. A secondary antibody or probe against labelled conjugate
antibody/aptamer is immobilized at control zone. Sample containing the analyte is applied to the sample application pad and it
subsequently migrates to the other parts of strip. At conjugate pad, target analyte is captured by the immobilized labelled antibody or
aptamer conjugate and results in the formation of labelled antibody conjugate/analyte complex. This complex now reaches at
nitrocellulose membrane and moves under capillary action. At test line, label antibody conjugate/analyte complex is captured by
another antibody which is primary to the analyte. Analyte becomes sandwiched between labelled and primary antibodies forming
labelled antibody conjugate/analyte/primary antibody complex. Excess labelled antibody conjugate will be captured at control zone by
secondary antibody. Buffer or excess solution goes to absorption pad. Intensity of colour at test line corresponds to the amo unt of
target analyte and is measured with an optical strip reader or visually inspected. Appearance of colour at control line ensures that a
strip is functioning properly. The double antibody sandwich format is used when testing for larger analytes with multiple antigenic
sites, such as LH, hCG, and HIV. In this case, less than an excess of sample analyte is desired so that some of the microspheres will
not be captured at the capture line, and will continue to flow toward the second line of immobilized antibodies, the control line. This
control line uses species-specific anti-immunoglobulin antibodies, specific for the conjugate antibodies on the microspheres.
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Schematic of sandwich format of LFA (a) Labelled lateral flow strip (b) When sample with target analyte is applied on sample
application pad, it flows over the strip under capillary action and colour appears at test and control lines. (c) When sample
without target analyte is applied on sample application pad, it flows and a colour appears only on test line.
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Competitive format
Such format suits best for low molecular weight compounds which cannot bind two antibodies simultaneously. Absence of
colour at test line is an indication for the presence of analyte while appearance of colour both at test and control lines indicates a
negative result. Competitive format has two layouts. In the first layout, solution containing target analyte is applied onto the sample
application pad and prefixed labelled biomolecule (antibody/ aptamer) conjugate gets hydrated and starts flowing with moving liquid.
Test line contains pre-immobilized antigen (same analyte to be detected) which binds specifically to label conjugate. Control line
contains pre-immobilized secondary antibody which has the ability to bind with labelled antibody conjugate. When liquid sample
reaches at the test line, pre-immobilized antigen will bind to the labelled conjugate in case target analyte in sample solution is absent
or present in such a low quantity that some site of labelled antibody conjugate were vacant. Antigen in the sample solution and the one
which is immobilized at test line of strip compete to bind with labelled conjugate. In another layout, labelled analyte conjugate is
dispensed at conjugate pad while a primary antibody to analyte is dispensed at test line. After application of analyte solution a
competition takes place between analyte and labelled analyte to bind with primary antibody at test line. Recently, a unique change was
introduced in conventional design of LFA by introducing a new line (antigen line) in between test and control lines for detection of C-
reactive protein (CRP) in serum samples. This format involves somehow a competition between analyte in solution and analyte pre-
dispensed on a new line. New line was formed by dispensing CRP antibody solution followed by CRP solution. In case of very low
concentration of CRP in sample, most of the labelled conjugate molecules will remain unreacted and migrate to antigen line and CRP
present at antigen line will capture these labelled conjugates and it will result in an intense colour at antigen line and rest of labelled
conjugate will move to control line and will produce relatively a light colour. In case of very high concentrations, most of CRP
molecules will be captured at test line and will be sandwiched in between labelled conjugate and prefixed antibody at test zone, this
complex will move and be captured by control line antibody. In this case very few labelled conjugate molecules will be retained at
antigen line. The lesser the colour at antigen line, the higher the concentration of analyte. This format can be tried for other clinical
and non-clinical analytes.
Schematic of competitive format of LFA (a) Labelled lateral flow strip (b) When a sample with target analyte is applied on
sample application pad, it flows through the strip and a colour appears on at control line. (c) when a sample without target
analyte is applied on sample application pad, it flows on the strip and colour appears on both test and control line.
Multiplex detection format

Multiplex detection format is used for detection of more than one target species and assay is performed over the strip
containing test lines equal to number of target species to be analysed. It is highly desirable to analyse multiple analytes simultaneously
under same set of conditions. Multiplex detection format is very useful in clinical diagnosis where multiple analytes which are inter-
dependent in deciding about the stage of a disease are to be detected. Lateral flow strips for this purpose can be built in various ways
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i.e. by increasing length and test lines on conventional strip, making other structures like stars or T-shapes. Shape of strip for LFA will
be dictated by number of target analytes. Miniaturized versions of LFA based on microarrays for multiplex detection of DNA
sequences have been reported to have several advantages such as less consumption of test reagents, requirement of lesser sample
volume and better sensitivity.
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DETECTION SYSTEMS
In case of gold nanoparticles or other colour producing labels, qualitative or semi-quantitative analysis can be done by visual
inspection of colours at test and control lines. The major advantage of visual inspection is rapid qualitative answer in ‘‘Yes’’ or
‘‘NO’’. Such quick replies about presence of an analyte in clinical analysis have very high importance. Such tests help doctors to
make an immediate decision near the patients in hospitals in situations where test results from central labs cannot be waited for
because of huge time consumption. But for quantification, optical strip readers are employed for measurement of the intensity of
colours produced at test and control lines of strip. This is achieved by inserting the strips into a strip reader and intensities are recorded
simultaneously by imaging software. Optical images of the strips can also be recorded with a camera and then processed by using a
suitable software. Procedure includes proper placement of strip under the camera and a controlled amount of light is thrown on the
areas to be observed. Such systems use monochromatic light and wavelength of light can be adjusted to get a good contrast among test
and control lines and background. In order to provide good quantitative and reproducible results, detection system should be sensitive
to different intensities of colours. Optical standards can be used to calibrate an optical reader device. Automated systems have
advantages over manual imaging and processing in terms of time consumption, interpretation of results and adjustment of variables.
In case of fluorescent labels, a fluorescence strip reader is used to record fluorescence intensity of test and control lines. Fluorescence
brightness of test line increased with an increase in nitrated ceruloplasmin concentration in human serum when it was detected with a
fluorescence strip reader. A photoelectric sensor was also used for detection in LFA where colloidal gold is exposed to light emitting
diode and resulting photoelectrons are recorded. Chemiluminescence which results from reaction of enzyme and substrate is measured
as a response to amount of target analyte. Magnetic strip readers and electrochemical detectors are also reported as detection systems
in LFTS but they are not very common. Selection of detector is mainly determined by the label employed in analysis.
APPLICATIONS
Clinical analysis
A major part of LFA applications lies in clinical analysis. It includes detection of a variety of clinical analytes in plasma,
serum, urine, cells, tissues and other biological samples.
RNA/DNA detection
MicroRNA was detected in cell lysate using a DNA-AuNP based LFA within a time of 20 min. DNA was quantified in
plasma by using dry reagent nucleic acid biosensor employing blue dye doped latex beads as a label. Detection was based on
hybridization between DNA conjugate and specific target DNA sequence in plasma. LFA was developed to identify nucleic acids by
using recognition properties of molecular beacons and optical properties of gold nanoparticles and very low detection limits were
achieved. Modified hairpin oligonucleotide with double target binding DNA sequence and gold nanoparticles was employed in LFA
for detection of single base mismatches in DNA by visual observation. Incorporation of double target DNA binding sequences into
loop of hairpin oligonucleotide has led an increase in the tendency of this probe to discriminate between perfect and single base
mismatches in DNA
Proteins and cells

Proteins serve as biomarkers for the uncovering of some diseases and their analysis has key prominence in clinical diagnosis.
Radioimmunoassay, protein chips, fluorescence, and other methods are used to detect low levels of proteins in biological matrices.
Disadvantages of these methods include disposal of radioactive substances, tiresome sample preparation steps, lavish instrumentation,
necessity of skilled analysers, washing and incubation procedures.
Concentration of thrombin protein in plasma samples was determined with high specificity using unique properties of
aptamers and gold nanoparticles in LFA. Cardiac marker cardiac troponin I is a protein, its concentration in bloodstream is very
important in determining and diagnosing acute myocardial infarction. In healthy people, concentration of cardiac troponin I is 20.4
pg/mL but as the AMI starts, the level of this protein marker rises with time and after few hours it reaches to its peak value 195.9
ng/mL. As central clinical laboratories consume much time in detection, rapid and sensitive methods are desired. Recently, LFA was
used for detection of human pluripotent stem cells employing gold nanoparticles as a label and it was capable of detecting down to
10,000 cells by visual inspection and 7000 cells by a strip reader. LFA based strip was prepared by combining molecular recognition
properties of aptamers and optical properties of gold for detection of cancer cells. Ramos cells were chosen as model analyte for this
study. Visual limit of detection was down to 4000 Ramos cells while a strip reader was able to detect minimum 800 Ramos cell.
Other clinical analytes

Diagnosis which involves tests on blood serum is termed as serodiagnosis. It involves diagnosis of disease by detection of antibody
or antigen. LFA was used to detect Leptospira-specific immunoglobulin M (IgM) antibodies in human blood serum and reported
results were in good agreement with routinely used ELISA. Human Brucellosis was diagnosed by LFA detection of brucella specific
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IgM antibodies in sera. Antibodies to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae were detected using LFA for
classification of leprosy patients and results showed good agreement with ELISA. Prostate specific antigen which is thought to be a
reliable marker for early diagnosis of prostate cancer was determined in human serum using gold nanoparticles as reporter and
electrochemical detection system [12].
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i. Immunochromatography along with google glass based for RDT reader capable of qualitative and quantitative measurements of
various lateral flow test and biomedical diagnostic test [16].
ii. Immunochromatography along with fluorescent bead provides new strategies to prevent the early stage transmission viruses in
humans during both seasonal outbreak and pandemics.
iii. Used to detect clenbuterol a compound in the urine using fluorescent Nano silica and visual detection limits.
iv. Used to determine ultra-small amounts of crustacean protein in processed foods which can lead to allergic reaction [8].
Foodborne pathogens and toxins

Botulinum neurotoxins are the most dangerous neurotoxins. They are produced by the Clostridium botulinum, which is spore
forming obligate anaerobe naturally, occurs in the soil. BoNts are divided into seven types. These toxins act to inhibit acetylcholine
release and result in paralysis and death. Highly sensitive LFA was designed to detect and differentiate between BoNT/A and B which
are known to be toxic and responsible for 80% of illnesses caused by milk and apple juice [17]. Corn, feedstuff and wheat were
screened for simultaneous detection of mycotoxins, zearalenone and fumonisin B1 by using a colloidal gold lateral flow strip. The
results were in good agreement with ELISA and LC-MS (Liquid chromatography-tandem mass spectrometry) [18].
Pesticides
Pesticides represent a wide class of chemicals including organic compounds which are volatile, semi-volatile or non-volatile
in their nature. Some inorganic compounds and organometallics are also used as pesticides but such instances are infrequent.
Pesticides have extensive applications in the agriculture division to grow crops and different food material. Through food chain, these
pesticides find their way to human body and wild animals. Two LFA strips for simultaneous detection of carbofuran and triazophos in
water samples were developed based on an immunogold conjugate. Unlike other strips, they contained two test lines and one control
line. Total analysis time was 10 min. Organophosphorus pesticides can be detected by using an indirect method. Their exposure results
in an increase of the total amount of phosphorylated cholinesterase which can be a biomarker to detect and quantify these pesticides.
Immunochromatographic strip coupled with disposable screen printed electrode was used to quantify this enzyme in in vitro red blood
cells and it can detect low up to 0.02 nM within a small period of time. A typical format of LFA was used for detection of paraoxon
methyl using Fe3O4 aggregates as a label and fluorescence strip reader as a detector.
Toxic pollutants
Bisphenol A (BPA) has wide applications in industry for preparation of epoxy resin, polycarbonate bottles, and also as a
flame retardant. It has been stated that Bisphenol A belongs to endocrine disrupting compounds and placed in watch list for further
review. Several reports have indicated involvement of Bisphenol A in reducing fertility and sperm quality in fishes. A simple and
rapid method based on LFA was designed for the detection of BPA in water and results showed better sensitivity compared to GCMS
(Gas chromatography Mass spectrometry) and LCMS (Liquid Chromatography Mass Spctrometry). Moreover, this method has
advantages of short analysis time, one step and on spot detection. A lateral flow strip method based on colloidal gold tag as a label was
used to analyze TNT in real samples and it was able to detect down to 1 mg/mL. Ractopamine which was used as feed additive in
livestock can be toxic to humans, LFA was successfully developed for its quantification in swine urine [12].
Heavy metals
Heavy metal pollution is the biggest concern to safety of human environment. Various environmental and health agencies
have regulated maximum allowable limits of metals in water, air and food stuff. Analytical techniques used for detection of heavy
metals are atomic absorption spectroscopy, inductively couple plasma mass spectrometry, and inductively coupled plasma optical
emission spectroscopy. These techniques cannot be utilized as rapid Point of Care testing because of large sized instruments, need for
expert personnel, and complex sample preparation.
A simple, sensitive and rapid visual detection of Hg2+ ions in aqueous solution was achieved by using gold nanoparticles as
reporter in LFA for coordination events of Hg2+between thymine rich hairpin oligonucleotide and digoxin labelled DNA probes which
was complementary to a part of hairpin oligonucleotide [19]. An immunochromatographic assay (ICA) was used to detect and quantify
chromium ions in water and serum samples using gold nanoparticles as tracers in a competitive format. Very low limit of detections
was got by visual and quantitative inspection and the strip was stable for 12 weeks at 37 oC without substantial loss of performance
[20]. Cd-EDTA-BSA-AuNP (cadmium-ethylenediaminetetraacetic acid-bovine serum albumin-gold nanoparticles) based LFA was
used to detect Cd2+ ions in tap and drinking waters and it resulted in 0.1 ppb detection limit which was so far better than any paper
based metal sensors [21].
Microfluidic devices for Point of Care diagnosis

Concept of POC (Point Of Care) testing has led to development of a variety of microfluidic devices. They can be divided
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based on their working principles, that is capillary driven (include LFA strips), pressure driven, centrifugal, electrokinetic and
acoustic. These devices have been reviewed in detail with an emphasis on working principle, market requirement, strengths and
limitations. A recent article extensively reviews applications of microfluidic devices for biomarker analysis [22]. After
polydimethylsiloxane, paper based microfluidics has got attention in recent years. Paper is a very cheap, abundant, light weight, thin
and flexible material and its main component is cellulose fibre which has already shown potential for diagnostics in LFA. Paper based
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Point Of Care devices have unique advantages of easy patterning, movement of fluid by capillary action, requirement of less volume
of sample and ease in disposing paper after testing.
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But they have disadvantage of varying sensitivity and selectivity. Several review articles covering both development and use
of paper based devices can be found in the literature [14, 23]. LFA strips are creating remarkable market share and despite their
limitations, they are most successfully commercially developed POC devices. Although some of other microfluidic devices have been
commercialized, main focus remained up to demonstrations. Huge initial investment and providing solution to all problems that might
occur are main hurdles in bringing these devices to consumers [24].
Human Chorionic Gonadotrophin (hCG) is a hormone secreted in pregnancy. It is excreted in urine of pregnant women.
Detection of this hormone in urine or serum is an easy method of diagnosis of pregnancy. Pregnancy detection kits containing a strip
or card impregnated with anti-hCG globulin are readily available. The test result can be obtained within 3 min by adding serum or
urine to the sample port [14, 25].
DEVELOPMENT POSSIBILITIES
The possibility of creating a truly quantitative test can be done by using the same format of lateral flow tests and dyeing the
solid support with a fluorescent dye. The amount of antibody bound at the capture line can be precisely quantified using a fluorimeter
if the spectral properties of the dyed microspheres to which the antibodies are conjugated are known. The currently existing all lateral
flow tests would provide all benefits to this and becomes theoretically a truly quantitative assay. By placing multiple lines of captured
antibodies on the membrane, for an each different analyte, an individual can develop a single test for more than one analytes. An exact
or obvious application for this is to create drugs of abuse test panel. Biosite’s ‘Triage’ is based on this pattern. This principle
diagnostically could be used for panels of which all multiple analytes can be tested i.e. immune diseases, allergies or multiple
chemical sensitivity disorder. As the technology involved in preparing these tests continues to be developing and progressing, it is
possible to combine both of these ideas, and to make a low-cost, rapid quantitative diagnostic assay for these multiple analytes [26].
Another promising possibility of this technology is in the field of environmental sciences which provides an opportunity to develop
rapid & reliable tests that can be performed in the fields of water pollution to plant disease. As these diagnostic tests must often be
performed in harsh environments lateral flow test format is unique for which, proper preparation, foil pouching and no refrigeration or
special handling is required. As scope in the field of molecular genetics continues to expand rapidly, the focus in using a simple
format for detecting various genetic markers, DNA and RNA related disease infectious pathogens is increasing. The core principle
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behind this type of test is the ability of a ligand from solution to bind with a solid support that can be performed on genetic material as
well as proteins, making this application of the technology in this field almost a limitless. Immunochromatography with protein coated
microspheres (Proactive streptavidin) allows in optimizing direct attachment of antibody.
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In this way, a series of tests can be developed rather quickly, using the same solid support, membrane and housing etc. [8].
Immunochromatography detection sensitivity is increased by replacing gold colloidal platinum and newly developed silver
amplification technology amplifies particles more than 100 times quickly and increase sensitivity. This amplified
immunochromatographic system is expected to be more useful for specimen under the condition liable to cause a false negative result
[26]. The human immunodeficiency virus (HIV) pandemic has become one of the greatest infectious disease threats to human health
and social stability that the world has ever faced. Every year around 6 lakhs new born children will get infected with HIV worldwide.
A pregnant woman with HIV infection has an approximately 30% chance of passing the virus to her born baby, prevention of parent to
child transmission is a specific programme that provides a comprehensive family centred spectrum of support and clinical services
along with other public health initiatives to prevent the transmission of HIV from parent to baby. So for this the sensitivity and
specificity of an Immunochromatography tests was 100% as compared to ELISA [8].
CONCLUSION
In last few years, more research focused on the use of LFA for detection of clinical and non-clinical analytes. LFA has
advantages of simple test procedure; requirement of low sample volume, fast analysis, no need for expert personnel and low cost of
operation. Integration of the nanotechnology into LFTS biosensors has resulted in enhanced signal to noise ratio, reduced analysis
time and simultaneous analysis of multiple analytes. Colloidal gold conjugation with biomolecules has provided an excellent platform
for detection of a variety of target analytes.
No doubt, the LFA strips have a broad range of applications in clinical and non-clinical analysis but several flaws have been
indicated by researchers which include poor reproducibility and less sensitivity toward high analyte concentrations. Most of LFAs
give qualitative or semi-quantitative results which can be observed by naked eyes. Conventional LFA are normally qualitative and
give answers in yes or no. A good LFA biosensor can be recognized by such figures of merit: biocompatibility, high specificity, high
sensitivity, rapidity of analysis, reproducibility/precision of results, wide working range of analysis, accuracy of analysis, high
throughput, compactness, low cost, simplicity of operation, portability, flexibility in configuration, possibility of miniaturization,
potential of mass production and on-site detection.
REFERENCES
1. P.C Kamboj. Pharmaceutical Analysis II (Instrumental Methods), 1 st edition, Vallabh Publications, chapter 4, Chromatographic
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Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: 2231-6876

IMMUNOCHROMATOGRAPHY: FORMATS AND APPLICATIONS

Cherian Sebastian 1*, Helen William2

Ion Exchange Chromatography

Molecular Exclusion Chromatography

IMMUNOCHROMATOGRAPHY ASSAY PRINCIPLE

LATERAL FLOW ASSAY VERSUS IMMUNOCHROMATOGRAPHY

This approach allows for:

WORKING OF IMMUNOCHROMATOGRAPHIC ASSAYS

Solubilisation of detector molecules

than allowing flooding or surface flow.

Conjugate or reagent pad

iii Processing Methods

the rearrangement and activity of proteins on the membrane.

characteristics in that period.

Availability and Choice:

Wick or waste reservoir

Magnetic particles and aggregates

Fluorescent and luminescent materials

than gold nanoparticles.

Multiplex detection format

Proteins and cells

Other clinical analytes

Foodborne pathogens and toxins

Microfluidic devices for Point of Care diagnosis

of lipoprotein (a) in plasma). Clin. Chem, 1993; 39: 619-624.

Food Control 31 (2013); 180–188.

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