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Lateral Flow Immunoassays

 

 

Point-of-Care (POC) diagnostic applications have been the focus of many discussions, conferences and meetings over the last 20 years. Companies have been working diligently to make products that are robust (reproducible), accurate and portable to the near-patient settings. One such type of assay which has had some success with this concept is the lateral flow immunoassay test (also known as the immunochromatography assay, or strip test). Like many great ideas, lateral flow immunoassays take an intellectually sophisticated technology and simplify it, allowing almost anyone to be able to perform the test.

Lateral flow assays are uncomplicated, intended to detect the presence (or absence) of a target analyte in sample (matrix) without the need for specialized or costly equipment (although, some lab based applications utilize reading equipment, particularly where a fluorescently tagged analyte has been used).

The basic principle of the lateral flow immunoassay was first described in the literature 20 years before the first real commercial test became available (a home pregnancy test launched in 1988). Since then, the technology has been used to develop a plethora of assays for clinical, veterinary, environmental, agricultural, bio-defensive and food-born pathogen screening applications. Strip assays are copiously adaptable and as such are commercially available for an extensive range of analytes including blood protein biomarkers, mycotoxins, viral and bacterial pathogens, as well as a whole range of nucleic acid detection products. These exemplify the vast range of products which this technology can be applied to.

The Technology

Lateral flow immunoassays are essentially immunoassays which have been adapted to operate along a single axis to suit the test strip format. There are a number of variations of the technology that have been developed into commercial products, but they all operate using the same basic concept.

lateral flow

The technology is based on a series of capillary beds, such as pieces of porous paper or polymer. Each of these elements has the capacity to transport fluid (e.g. blood) precipitately.

A typical lateral flow assay test strip consist of the following components:

1. Sample pad

An adsorbent pad onto which the test sample is applied.  This acts as a sponge and holds an excess of sample fluid.

2. Conjugate or reagent pad 

Once the sample pad is saturated the fluid migrates to the conjugate pad which contains antibodies specific to the target analyte conjugated to coloured particles (usually gold nanoparticles, or latex microspheres but in some instances fluorescent labels are used). When the sample fluid dissipates the matrix, it also dissolves the particles and in one combined, conveying action, the sample and conjugate mix flow through the porous structure. In this way, the analyte binds to the particles while migrating further through the third capillary bed.

3. Reaction membrane

This is typically a hydrophobic nitrocellulose or cellulose acetate membrane onto which anti-target analyte antibodies are immobilised in a line that crosses the membrane to act as a capture zone or test line (a control zone will also be present, containing antibodies specific for the conjugate antibodies). 

This material has one or more areas (often called strips) where a third molecule has been immobilized by the manufacturer. By the time the sample-conjugate mix reaches these strips, the 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 color. 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. 

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.

4. Wick or waste reservoir

A further absorbent pad designed to draw the sample across the reaction membrane by capillary action and collect it. After passing these reaction zones the fluid enters the final porous material, the wick, which simply acts as a waste container.

To learn more about lateral flow assays please watch the following webinar: 

 

Nanoparticles for lateral flow assays

The in-vitro diagnostic market requires antibodies to be linked to nanoparticles. Gold nanoparticles are attractive for the development of reagents for diagnostic devices (lateral flow assays) because of their intense ruby red colour. The production of nanoparticle antibody conjugates is challenging because of instability, and numerous parameters need to be optimised. At least 60 different batches are usually made on each project, often requiring specialist knowledge. 

Furthermore, the IVD market is tightly regulated by organisations such as the USA Food and Drug Administration. Major criteria for satisfying such organisations are quality and reproducibility of the gold conjugates. Often as a result of batch-to-batch variability, nanoparticle-conjugates are returned to R&D for further optimisation, thus delaying product launch.

 

 

The Solution InnovaCoat® GOLD nanoparticle conjugation kits

InnovaCoat® GOLD products are ‘conjugation friendly’ nanoparticles with a proprietary surface coat that greatly enhances gold stability and permits easy covalent attachment of a variety of molecules, including antibodies, analytes and other biomolecules. The conjugation reaction is initiated simply by adding a solution of antibody to the freeze-dried powder; the hands-on time is 2 minutes and the conjugate is ready to use within 15 minutes.

In the case of immuno-gold conjugates, the antibody can be attached irreversibly without the need for extensive trials at different values of pH and/or salt concentration, as is typical of traditional ‘passive’ binding methods.

Please view the video below for a visual representation of InnovaCoat® GOLD.

 

InnovaCoat® GOLD conjugates can be used in a variety of assays including lateral flow tests; as well as avoiding the optimisation steps, InnovaCoat® GOLD conjugates demonstrate enhanced sensitivity when compared to the traditional passive methods - please see comparative graph below.

lateral flow

Competitive lateral flow assay comparing antibodies conjugated to 40nm gold particles by a traditional passive method against those conjugated covalently using InnovaCoat® GOLD.

Find out more on our InnovaCoat® GOLD webpage.

 

by InnovaBiosciences 

 

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