Lateral Flow Immunoassays
This lateral flow application guide will cover the following:
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.
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.
Figure 1. Typical Lateral Flow Assay
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:
Gold and latex are the most commonly used particles for lateral flow assays and there are advantages to using both systems. Successfully conjugating an antibody or other protein to either can be time consuming and challenging, so we have developed solutions to make these steps easier. We sell specially treated latex and coated gold conjugation kits that offer a one-step conjugation reaction. We also offer colloidal gold for use traditional conjugation techniques for the more experienced user, as well as pre-conjugated gold secondary reagents.
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. Many different conjugates are usually made on each project, and this type of optimization often requires 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.
InnovaCoat® GOLD nanoparticle conjugation kits
Our InnovaCoat® GOLD conjugation kits contain ‘conjugation friendly’ nanoparticles that have a proprietary surface coat which 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.
Take a look at our protocol video demonstration to see how easy-to-use the kits are:
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 - see Figure 2.
|Figure 3. Lateral flow assay of our 40nm InnovaCoat® GOLD nanoparticles conjugated to Goat-Anti-Rabbit titrated against Rabbit IgG. The signal intensity of the T-line clearly changes in proportion to the antigen concentration.||
Figure 4. Quantification of T-line signal of the lateral flow assay displayed in figure 3 (left) with a lateral flow reader.
|Figure 5. Our 40nm InnovaCoat® GOLD nanoparticles conjugated to Goat-Anti-Rabbit are run on a sandwich lateral flow assay in the presence/absence of Rabbit IgG. The presence of an antigen can be visually assessed by a signal on the T-line on a lateral flow assay. The C-line confirms that an assay has run properly and should always be visible.|
Gold nanoparticle conjugates
We also offer pre-conjugated nanoparticles manufactured using our InnovaCoat GOLD technology. Take a look at the full range of pre-conjugated gold nanoparticles here.
Uncoated gold nanoparticles are also used for lateral flow immunoassays although this requires passive adsoprtion of the antibody onto the surface of the uncoated nanoparticle which is technically complex and require expertise in passive adsorption techniques. Take a look at our ultra-high quality colloidal gold here.
Please take a look at the video below for more information about our range of gold nanoparticles:
Latex beads are also commonly used particles for lateral flow assays. As with gold nanoparticles, latex beads are conjugated to the detection antibody and upon detection of present analyte, produce a clear signal which is detectable by eye or using a lateral flow strip reader.
One of the attractive properties of using latex for in-vitro diagnostics is the availability of the beads in vibrant different colors, therefore allowing for the simultaneous detection of different analytes (figure 6.)
Figure 6. Multiplexed Latex. The three colours of latex each conjugated to a different antibody or protein and forming a line either by a direct binding event (red and black latex) or a sandwich assay binding event (blue latex). The three colours of latex demonstrate no aggregation or background staining.
However the conjugation of latex beads to the antibody or protein of choice can be difficult and time consuming. Traditional methods, whether passive or covalent, are technically complex and can waste a lot of material.
Traditional passive conjugation requires the adsorption of the antibody onto the surface of the latex, often extensive optimisation is required to find the right pH meaning wasted materials and wasted time. With covalent conjugation, aggregation of the latex is a big issue, and even with optimised protocols harsh methodologies such as vortexing and sonication are required to minimise aggregation.
That’s why we developed our LATEX conjugation kits, containing everything required for conjugating your antibody to our specially treated latex beads for use in your lateral flow assay.
Our kits allow you to conjugate your antibody to either red, blue or black latex beads with only 3 minutes hands-on time and conjugates are ready to use in 35 minutes.
So why use our latex conjugation kits over traditional methods?
- Less antibody is required – up to 10x less
- No extensive trials to find optimal pH
- No optimisation to reduce aggregation
- Fewer steps involved, less time wasted!
Find out more about our latex conjugation kits for lateral flow here.
If you would like anymore information about any of the above or have a technical question, we have an expert technical team who will be happy to help! Get in touch below: