immunochromatography: formats and … · please cite this article in press as cherian sebastian et...

www.iajpr.com

Pag

e64

00

Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: 2231-6876

IMMUNOCHROMATOGRAPHY: FORMATS AND APPLICATIONS Cherian Sebastian

1*, Helen William

2

1Scholar, College of Pharmaceutical Sciences, Govt. Medical College, Kottayam, Kerala, India

2Assistant Professor, College of Pharmaceutical Sciences, Govt. Medical College, Kottayam, Kerala, India

Corresponding author

Cherian Sebastian

Scholar,

College of Pharmaceutical Sciences,

Govt. Medical College,

Kottayam, Kerala, India

[email protected]

+917736596951

Copy right © 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical

Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ARTICLE INFO ABSTRACT

Article history

Received 13/07/2016

Available online

12/08/2016

Keywords

Immunochromatography,

Advantages and

Disadvantages,

Working,

Formats,

Applications.

Immunochromatography is a combination of chromatography and immunoassay. In this

technique, the antigen-antibody reaction which occurs on a membrane is used to determine

the target analyte in the sample. For this the specific biomolecule against antigen or antibody

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

analyte. The method has widespread application in detecting large kind of molecules

including poisonous substances, pathogens in their smallest levels, hormones, etc. By the

application of this principle, presence of disease causing organism in the body can be

detected in early stages at a relatively lesser cost. This review article provide an overview on

principle, working, formats, advantages, disadvantages and some of the important

applications of immunochromatography.

Please cite this article in press as Cherian Sebastian et al. Immunochromatography: Formats and Applications .Indo American

Journal of Pharmaceutical Research.2016:6(07).

mailto:[email protected]

www.iajpr.com

Pag

e64

01

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 PH.

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

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].

www.iajpr.com

Pag

e64

02


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

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.

www.iajpr.com

Pag

e64

03


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

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].

www.iajpr.com

Pag

e64

04


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).

www.iajpr.com

Pag

e64

05


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].

www.iajpr.com

Pag

e64

06


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

than allowing flooding or surface flow.

www.iajpr.com

Pag

e64

07


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

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.

www.iajpr.com

Pag

e64

08


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

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

characteristics in that period.

www.iajpr.com

Pag

e64

09


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 107

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.

www.iajpr.com

Pag

e64

10


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

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

than gold nanoparticles.

www.iajpr.com

Pag

e64

11


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 amount 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.

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.

www.iajpr.com

Pag

e64

12


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

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.

www.iajpr.com

Pag

e64

13


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

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].

www.iajpr.com

Pag

e64

14


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 37oC 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

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

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.

www.iajpr.com

Pag

e64

15


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

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.

www.iajpr.com

Pag

e64

16


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), 1st edition, Vallabh Publications, chapter 4, Chromatographic

separarion-An introduction; p. 63-69

2. Uhlén M (2008). Affinity as a tool in life science. Biotechniques 44 (5): 649–654. doi:10.2144/000112803. PMID 18474040.

3. Kellner R, Mermet JM, Otto M, Widmer HM. Analytical Chemistry.New York: Wiley-VCH. 1998; p405-42911.

4. Findlay JW, Smith WC, Lee JW, Nordblom GD, et al. J Pharm. Biomed. Anal. 2000; 21: 1249-1273.

5. Fiets WE, Blankenstein MA, Struikmans H, Ruitenberg HM, et al. Int. J. Biol. Markers. 2002; 17: 24-32.

6. Pieniaszek HJ, Davidson AF, Walton HL, Pinto DJ, et al. J .Pharm . Biomed. Anal. 2003; 30: 1441-1449.

1. 7. Ananthanarayanan and Panicker’s Text book of microbiology,7th

edition, Oreint Longman Private limited,Part-II,13Antigen

Antibody Reactions; p105-108.

7. B. Ravishankar, R. Rajesh, E. Venkatanagaraju, G. Divakar, T. Shreeshail, Y. Nehalatha Reddy, Over view on development and

applications of immunochromatography. World journal of pharmacy and pharmaceutical science. Volume 4, Issue 12, p.344-349

8. M.M. Rhemrev-Boom, M. Yates , M. Rudolph , M. Raedts, (Immuno)affinity chromatography: a versatile tool for fast and

selective purification, concentration, isolation and analysis. Journal of Pharmaceutical and Biomedical Analysis 24 (2001):825–

833

9. 9025 Technology Dr.Fishers, IN 46038-2886 Bangs laboratories Inc. Technote 303: Lateral flow tests, 800.387.0672 •

317.570.7020.

10. Boris B. Dzantiev , Nadezhda A. Byzova, Alexandr E. Urusov, Anatoly V. Zherdev, Immunochromatographic methods in food

analysis. A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, 119071 Moscow,

Russia,p:400-432

11. Muhammad Sajid, Abdel-Nasser Kawde, Muhammad Daud, Designs, formats and applications of lateral flow assay: A literature

review”. Journal of Saudi Chemical Society (2015) 19:689–705.

12. Marja E. Koivunen, Richard L. Krogsrud, Labmedicine Review ”Principles of Immunochemical

Techniques Used in Clinical Laboratories”, August 2006,Volume 37, Number 8,p:490-491

13. A.K. Yetisen, M.S. Akram, C.R. Lowe, Paper-based microfluidic point-of-care diagnostic devices, Lab Chip 13 (2013) 2210–

2251.

14. R.C. Wong, H.Y. Tse (eds.), Lateral Flow Immunoassay, DOI 10.1007/978-1-59745-240-3_1, Humana Press, New York, NY

2009.

15. Lou S, Patel C, Ching S and Gordon J. (One step competitive immunochromatographic assay for semiquantitative determination

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

16. K.H. Ching, A. Lin, J.A. McGarvey, L.H. Stanker, R. Hnasko, Rapid and selective detection of botulinum neurotoxin serotype-A

and -B with a single immunochromatographic test strip, J. Immunol. Methods 380 (2012) 23–29.

17. Y.-K. Wang, Y.-B. Shi, Q. Zou, J.-H. Sun, Z.-F. Chen, H. Wang, et al, Development of a rapid and simultaneous

immunochromatographic assay for the determination of zearalenone and fumonisin B1 in corn, wheat and feedstuff samples,

Food Control 31 (2013); 180–188.

www.iajpr.com

Pag

e64

17


18. Y. He, X. Zhang, K. Zeng, S. Zhang, M. Baloda, A.S. Gurung, et al, Biosensors and bioelectronics visual detection of Hg2+

in

aqueous solution using gold nanoparticles and thymine-rich hairpin DNA probes, Biosens. Bioelectron. 26 (2011) 4464–4470.

19. X. Liu, J. Xiang, Y. Tang, X. Zhang, Q. Fu, J. Zou, et al, Colloidal gold nanoparticle probe-based immunochromatographic assay

for the rapid detection of chromium ions in water and serum samples, Anal. Chim. Acta 745 (2012): 99–105.

20. A.M. Lopez Marzo, J. Pons, D.A. Blake, A. Merkoc I , High sensitive gold-nanoparticle based lateral flow Immunodevice for

Cd2+

detection in drinking waters, Biosens. Bioelectron. 47 (2013) 190–198.

21. S. Nahavandi, S. Baratchi, R. Soffe, S.-Y. Tang, S. Nahavandi, A. Mitchell, et al, Microfluidic platforms for biomarker analysis,

Lab Chip 14 (2014) 1496–1514.

22. W.K. Tomazelli Coltro, C.-M. Cheng, E. Carrilho, D.P. de Jesus, Recent advances in low-cost microfluidic platforms for

diagnostic applications, Electrophoresis 35 (2014):2309–2324.

23. D. Mark, S. Haeberle, G. Roth, F. von Stetten, R. Zengerle, Microfluidic lab-on-a-chip platforms: requirements, characteristics

and applications, Chem. Soc. Rev. 39 (2010):1153–1182.

24. Wilcox AJ, Baird DD, Weinberg CR (1999), Time of implantation of the conceptus and loss of pregnancy, New England Journal

of Medicine 340 (23): 1796-1799.

25. Mikinaga M, Junuchi K, Hiroyuki C, Kentaro N, Takayoshi O. (Development of highly sensitive Immunochromatographic

detection kit for seasonal influenza virus using silver amplification). Fujifilm research and development, 2012; 57: 8-11.

54878478451160723

immunochromatography: formats and … · please cite this article in press as cherian sebastian et...

Documents