Lateral Flow Assay

While rapid assay methods have made a major impact on a variety of diagnostic testing over the last twenty years only a handful of development can make the claim to have taken testing out of the laboratory.

One that can, and is in widespread use as a result, is the lateral flow immunoassay test, also known as the immunochromatography assay, or strip test. Like many of the best ideas, lateral flow immunoassays take clever and sophisticated technology and turn it into something so simple to operate that almost anyone can use it.

The Technology

The basic technology that underlies lateral flow immunoassays was first described in the 1960s, but the first real commercial application was Unipath’s Clearview home pregnancy test launched in 1988. Since then, this technology has been employed to develop a wide and ever-growing range of assays for clinical, veterinary, agricultural, food industry, bio-defence and environmental applications.

Strip assays are extremely versatile and are available for an enormous range of analytes from blood proteins to mycotoxins and from viral pathogens to bacterial toxins. Assays has even been developed for wine producers to assess the amount of botrytis rot in newly harvested grapes as well as for use in the clinical lab identifying cardiac markers. This shows the vast range that this technology can be applied too.

Lateral flow immunoassays are essentially immunoassays 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 one being Vertical Flow Technology, but they all operate using the same basic principle.

Figure 1. Advantages of lateral flow assays.

How Does a Lateral Flow Assay Work?

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

Sample pad – an adsorbent pad onto which the test sample is applied.

Conjugate or reagent pad – this contains antibodies specific to the target analyte conjugated to coloured particles (usually colloidal gold nanoparticles, or latex microspheres).

Reaction membrane – typically a nitrocellulose or cellulose acetate membrane onto which anti-target analyte antibodies are immobilized 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).

Wick or waste reservoir – a further absorbent pad designed to draw the sample across the reaction membrane by capillary action and collect it.

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.

Figure 2. Principle of lateral flow assay.

Cytodiagnostics manufactures a full product line of gold nanoparticles (colloidal gold) for use in a variety of lateral flow assays. Our diverse product line of different type of nanoparticles offers you products with a narrow size distribution (CV of less than 12%), exceptional adsorption and conjugation properties and with greater than 95% spherical particles. In addition, our batch to batch variability is extremely low (+/- 2nm), which assures that you our customer will always end up with a product within the specified size range that you ordered.

The high shape uniformity of our colloidal gold will minimize the variability within your assay by e.g. allowing control over the available surface area while absorbing or covalently conjugating proteins to our gold nanoparticles. It will also ensure a more uniform flow rate across your membrane for improved reproducibility and overall results.

Background

Vertical flow immunoassays rely on the same basic principles as the more common lateral flow immunoassay format with some modifications. The most apparent difference between the two methods being the vertical and lateral flow of fluid. However, vertical flow technology has several advantages over traditional lateral flow assays with the most significant being the reduced assay time (<5 minutes), table I.

Vertical flow immunoassays can be used for rapid detection of an antigen(s) in a sample of interest. Samples can come from a wide range of application areas such as clinical, veterinary, agricultural, food, bio-defence and environmental industries. Detection sensitivity is generally in the lower nanogram per ml range even in complex sample matrices.

Table I. Features and benefit of rapid vertical flow technology compared to lateral flow and other rapid flow type tests.

How does a Vertical Flow Assay Work

As with lateral flow, vertical flow immunoassays rely on the immobilization of a capture antibody on a reagent pad to which the sample of interest (with or without antigen to be detected) is applied. Detection of the bound antigen is subsequently achieved through the binding of an antigen specific antibody gold conjugate. This step completes a sandwich consisting of a capture antibody, an antigen and finally the gold conjugate and results in a direct and permanent visually detectable red coloured dot indicating the presence of the antigen, figure 1.

Of interest is that alternative colour of detection can easily be achieved by using a different type of nanoparticle conjugate probe for detection. One example being antibody conjugated gold nanourchins, which will yield a blue coloured dot upon positive detection in the assay.

Figure 1. Illustration of the appearance of the Miriad™ vertical flow cartridge after applying a gold conjugate (A) or a gold nanourchin conjugate (B) when target is present (A, B) and not present (C) in the sample analyzed.

Multiplexing Capabilities in Vertical Flow Immunoassays
Vertical flow technology also allows for easy multiplexing. In the Miriad™ vertical flow test cartridges distributed by Cytodiagnostics Inc., the presence of 4 targets can be evaluated simultaneously in a single sample in 5 minutes or less.

Multiplexing is easily achieved by spotting capture antibodies against different antigens at pre-determined locations and/or patterns on the membrane. For easier visualization this multiplexing can also be coupled with nanoparticle probes of different colours, e.g. using a gold conjugate and a gold nanourchin conjugate with red and blue detection colours, respectively.

Figure 2. Multiplex detection in a vertical flow immunoassay. Left image shows positive detection of 2 out of 3 antigens (red dots) and the control. The right image shows positive detection of two different antigens using two nanoparticle probes with different colours (red: gold nanoparticles, blue: gold nanourchins.

Passive adsorption is a method employed in the conjugation of proteins and antibodies to gold nanoparticles, a fundamental step in developing lateral flow assays (LFAs). This technique leverages the natural affinity between the gold surface and certain biomolecules, allowing them to adsorb onto the nanoparticles without the need for complex chemical reactions.

In lateral flow assays, the conjugation of biomolecules to gold nanoparticles is crucial for the detection and quantification of target analytes. Passive adsorption is particularly valued in this context for its simplicity and effectiveness. The process ensures that the biomolecules retain their structural integrity and functional properties, which are essential for the assay’s sensitivity and specificity.

If you are conjugating antibodies to our gold nanoparticles, we also offer Conjugation QC Lateral Flow Dipsticks to validate loading of your antibody onto the gold nanoparticle surface.

Covalent conjugation is a technique used to attach biomolecules, such as proteins, antibodies, or nucleic acids, to gold nanoparticles through stable covalent bonds. This method involves creating a permanent link between the biomolecule and the nanoparticle, ensuring a strong and lasting attachment that is crucial for the stability and functionality of lateral flow assays (LFAs).

This type of surface chemistry offers a high degree of control over the orientation and density of biomolecules on the nanoparticle surface. In addition, surface loading of PEG further enhances the conjugate’s stability and is highly favourable for use in testing targets found in complex sample matrices.

Nucleic acid conjugation kits are specialized tools designed for attaching oligonucleotides and aptamers to gold nanoparticles. This process is pivotal in the development of lateral flow assays (LFAs) that target specific genetic markers or molecular structures.

The utility of these kits lies in their ability to facilitate a stable and precise binding of nucleic acids to the nanoparticles. This precision is crucial for assays that depend on the accurate recognition and binding of specific nucleic acid sequences. In these assays, the correct orientation and presentation of the nucleic acids on the nanoparticle surface are vital for effective target detection.

NOTE: These kits are to be used with either 5′ or 3′ Thiolated Oligonucleotides. It is important that all oligonucleotides are deprotected for proper conjugation with the OligoREADY and AptamerREADY conjugation kits. Please follow this guide if you require assistance.