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Lateral Flow Assay Development

Creative diagnostics’ lateral flow assay (LFA) development service will take your assay development from initial idea, through to R&D and then transfer to trusted partners for bulk manufacturing.

Lateral flow tests based on the principles of immunochromatography exist for a wide array of target analytes for drugs of abuse tests, food safety tests, fertility tests, cardiac markers tests, infectious disease tests, and other tests important to human physiology. While the first tests presented qualitative results based on the presence or absence of a signal line, test design has progressed toward semi-quantitative and quantitative assays and the integration of hand-held readers.

1. Common Components of Lateral Flow Tests   >> See more at technical note 1

Sample pad - an adsorbent pad on which sample is dropped, impregnated with buffer salts and surfactants that make the sample suitable for interaction with the detection system.

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

The Membrane/Analytical Region - 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).

Absorbent Pad - a further absorbent pad designed to draw the sample across the reaction membrane by capillary action and collect it.

Plastic adhesive backing card - All components of the lateral flow assay are laminated to the backing material to provide rigidity and easy handling of the strip.

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 test and control zones.

2. Common Formats of Lateral Flow Tests   >> See more at technical note 2

There are several formats of Lateral Flow Tests chosen depending on the analyte.

2.1 Competitive Format Lateral Flow Assay

When the analyte is of low molecular mass and has only one epitope, i.e. a hapten, the competitive format LFA is used. Two layouts are possible:

• Antibody is sprayed at the test line, a mixture of sample analyte and labelled analyte is applied at the conjugate pad and the sample analyte and labelled analyte compete for binding sites on the antibody at the test line;

• An analyte-protein conjugate is sprayed at the test line, and a mixture of labelled antibody and sample analyte is applied at the conjugate pad, giving the sample analyte a head start for binding to the antibody.

In the competitive LFIA format the response is negatively correlated to the analyte concentration (i.e. more analyte present, less signal; no analyte gives the highest signal).

2.2 Sandwich Format Lateral Flow Assay

For large analytes with more than one antigenic site, the sandwich format is applicable. In this format the test line is prepared using primary antibody against the antigen (detection antibody). The conjugate pad contains a labelled, anti-analyte antibody. When sample solution containing target analyte is added into sample pad, target analyte and labeled antibody form a complex in the conjugation pad and migrate to absorbent pad. The target analyte-labeled antibody conjugate reacts with detection antibody on the test line and a sandwich is formed. Labeled antibody, which is free of analyte exceeding the test line, is bound to the sencond antibody immobilised on the control line. The response is directly proportional to the amount of analyte in the sample.

2.3 Nucleic Acid Lateral Flow (Immuno) Assay

The NALFIA set-ups are usually designed for testing the presence or absence of pathogens in food, feed or the environment. In the NALFIA set-ups, the analyte is an amplified double-stranded nucleic acid sequence (ds-amplicon) specific of the organism using primers with two different tags; recognition of the analyte is done by binding to a tag-specific antibody. In a typical layout developed for the detection of pathogenic bacteria the nucleic acid was amplified using PCR with two tagged primers. A ds-amplicon was obtained with one strand labelled with biotin and the other strand labelled with, e.g., fluorescein isothiocyanate or digoxigenine. A solution of antibodies raised against the tag was sprayed at the test line. The biotin will bind to the avidin-labelled nanoparticles and the other tag will bind to the antitag antibody, resulting in the coloured signal. The response is directly proportional to the amount of analyte.

3. Major Steps In LFA

3.1 Preparation of Antibody Against Target Analyte   >> See more at technical note 4

The heart of LFA is the antibodies, which need to be carefully designed with sufficient sensitivity, specificity, purity, and stability. It is very important to ensure a consistent antibody supply with proven affinity and specificity. The antibody must remain reactive after being adsorbed to a solid surface.

3.2 Preparation of Label   >> See more at technical note 3

Nanoparticles with specific optimal properties have been used as a tracer for LFA development. Gold nanoparticles, carbon nanoparticles, quantum dots, fluorescent material, up-converting phosphor, superparamagnetic nanoparticles are used as a label because of the signal amplification property to achieve high sensitivity and selectivity for target analysis in LFA. Colloidal gold is the most widely used label in commercial LFIA because of its high stability and low cost. Carbon and fluorescent labels, or enzymatic modification of the labels, are also used to improve the sensitivity of the assay. Carbon nanotubes have been shown to exhibit a limit of detection that is 10-fold lower than that of gold. Fluorescent nanoparticles such as quantum dots, time-resolved fluorescent nanoparticles have high signal to noise ratio, lower and quantitative detection are obtained. Superparamagnetic nanoparticles (MNPs) are new labelling materials to develop a LFA. They increase the senstitivity almost 10 to 100 times. They have low background noise, because the magnetic material does not exist usually in the environment or in the tested samples.

3.3 Labeling of Biorecognition Molecules   >> See more at technical note 4

The two operations, conjugating various types of antigens (or antibodies) to labels, such as Gold colloid QDs, MNPs, fluorescent labels, and immobilizing the antigens (or antibodies) to Nitrocelluose membrane affect to specificity and sensitivity of in vitro diagnostic, which are related to the quality of the product.

3.4 Assembling   >> See more at technical note 4

The basic processing steps for a cassette-based LFIA involve dispensing of reagents, immersion of materials into bulk solutions of reagents, drying of components, lamination of materials, cutting into strips, cassette assembly and packaging.

3.5 Optimizing   >> See more at technical note 4

Regardless of the manufacturing process used, the striping protocol for reagent application must be optimized. The optimization process includes choosing the appropriate antibody pair, conjugation conditions, sample pad and conjugate pad material and treatment, nitrocellulose membrane, test line concentration, wick pad material, running buffer, cassette, and sample volume. Optimization of your assay will provide you with the best possible performance at the lowest manufacturing cost.

3.6 Application of Sample and Obtaining Results

In the direct test, the presence of the test line indicates a positive result and the control line usually contains species-specific anti-immunoglobulin antibodies, specific for the antibody in the particular conjugate. In the case of small molecules with single antigenic determinants, which cannot bind to two antibodies simultaneously, competitive tests are used. In this type of test, the analyte blocks the binding sites on the antibodies on the test line, preventing their interactions with the coloured conjugate. Therefore, a positive result is indicated by the lack of signal in the test line, while the control line should be visible independently of the test result. Tests on which the control line does not appear should be deemed invalid and a fresh test should be run.

4. Device Reader    >> See more at technical note 5

Several reader devices have become commercially available and these can be grouped into fluorescence, magnetic particle, and colorimetric readers. In many cases, charge-coupled device (CCD)-based image systems and scanning systems are also utilized in these readers. Since fluorescence dyes and paramagnetic particle cannot be detected by the naked eye directly, such technologies immediately create demands for the development of readers for quantitative analysis. In order to avoid false negative and false positive results in colloidal gold- or latex bead-based assays, colorimetric scanning reader is performed by absorption and reflection of the light which are proportional to the overall density of colloidal gold particles of the band.

Many LFIAs are designed for use at point-of-care/need, providing cheap, rapid and easy tests desirable in many industries. However, regulatory bodies often require confirmation of results using an independent method. Therefore, LFIA are only suitable for primary screening at point-of-care/need. Because of their long shelf life and the fact that refrigeration is not required for storage, these tests are very well adapted for use in developing countries. As the visual result is usually clear and easily distinguished, no additional specific equipment is needed. Research is on-going to address some of the key weaknesses of LFAs, especially with respect to quantitative results. Data can be digitized using scanners or cameras with dedicated software, which will also allow the documentation of results. However, technological improvements will affect the cost of apparatus and the duration of analysis.

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