Lateral Flow Application

A lateral flow assay (LFA) is a simple and cost-effective diagnostic tool that is commonly used for the rapid detection of specific analytes in a sample. This type of assay is based on the principle of capillary flow, which refers to the movement of a liquid through a porous material under the influence of a pressure gradient. The key feature of a lateral flow assay is the flow of a liquid sample across a nitrocellulose strip that contains specific capture reagents. These reagents are designed to bind the target analyte and produce a visible signal that indicates the presence or absence of the target in the sample. 

A lateral flow assay (LFA) typically consists of the following components:



      1. Sample pad: This is where the sample is applied. It is usually made of nitrocellulose or cellulose and is designed to absorb and distribute the sample evenly across the test strip.
      2. Conjugate pad: This pad contains the reagents required for the assay, such as antibodies or enzymes, that will interact with the analyte in the sample.
      3. Nitrocellulose membrane: This layer acts as a filter to separate the analyte from the sample. The membrane is impregnated with capture antibodies that will bind to specific antigens in the sample.
      4. Test line: This line indicates the presence of the analyte being tested for. It appears as a colored band if the analyte is present in the sample.
      5. Control line: This line is usually present on the test strip and is used to indicate that the assay is working properly. It is usually a colored band that appears if the test is performed correctly and the reagents are still active.
      6. Absorbent pad: This pad is located at the end of the test strip and is used to absorb any excess fluid, ensuring that the test results are not compromised.

    Sandwich Assay

    The process of a lateral flow assay typically begins with the application of a liquid sample to the sample pad at one end of the test strip. The sample then wicks or flows laterally along the strip due to the capillary action of the nitrocellulose  membrane. As the sample moves along the strip, it encounters a capture reagent, commonly antibodies that are incorporated into the strip. If the target analyte is present in the sample, it will bind to the capture reagents and form a complex that is immobilized on the strip. 

    The next step in the assay is the detection of the target analyte. This is typically accomplished using a secondary detection reagent, such as a gold-antibody conjugate, that is specific for the target-capture complex. The antibody conjugate travels along the strip by capillary flow and binds to the target-capture complex, forming a visible signal that indicates the presence of the target analyte. The signal is usually a line or a spot that appears on the strip and is easily visible to the naked eye. 

    The interpretation of the results of a lateral flow assay is usually straightforward. If a visible signal is present on the strip, it indicates that the target analyte is present in the sample. If no signal is present, it indicates that the target analyte is not present in the sample. Some lateral flow assays also include a control line or spot that provides a visual indication of whether the test has been performed correctly. 

    The signal that appear can be read visually for qualitative or semi-quantitative assays or even with an intensity reader for full quantitation. Sandwich assays work best for large analytes, especially those with more than one binding site. In this format, the analyte should not be in excess of the binding sites on the conjugate, allowing unbound conjugate to continue to the control line.

    Sandwich Type Lateral Flow Assay

    Competitive Assay

    Alternatively, in competitive assays, the analyte of interest is conjugated to the nanoparticle. When the sample containing the analyte is added to the sample pad, the analyte's smaller size enables it to migrate faster through the porous membrane than the analyte-nanoparticle conjugate, blocking the available binding sites (Figure 3b-c). The analyte conjugate is therefore unable to bind to the test line, and is instead sequestered by the control line.
    In the absence of analyte in the sample, the analyte  conjugate is able to bind with both the test line and the control line (Figure 3d). In a competitive assay, a negative sample will show both a test and control line, and a positive sample yields only a control line (Figure 4). Competitive assays are particularly useful for smaller analytes which cannot bind to two recognition elements at once.

    Competitive Type Lateral Flow Assay


    Lateral Flow Optimization 

    In order to develop a working lateral flow assay, the components must be optimized for the analyte of interest. 

    Sample pad:
    1. Must render the sample suitable for the assay (e.g. by. filtering out disrupting and larger components of the sample matrix).
    Running buffer:
    1. In order to maximize specific binding and minimize non-specific binding, buffer components must be optimized for each unique analyte.
    2. Buffer pH, salt, protein and detergent content can be adjusted to optimize binding properties and sample flow through the membrane.
    Conjugate pad:
    1. May favour fast or slow release, but release must be consistent and reproducible over shelf life.
    2. Usually require pre-treatment by immersion in protein/surfactant solution to enable efficient conjugate release.
    3. Conjugate addition:
      1. immersion - less instrumentation required but less quantitative
      2. quantitative spraying by dispenser
    4. Conjugate: see separate section below.
    1. Pores should be large enough to allow the analyte-nanoparticle conjugate to flow through rapidly enough for quick detection, but  slowly enough to allow the analyte to interact with and bind to the test line. Typical pore sizes rant from 8 to 15 microns, yielding slower to faster wicking rates.
    2. Various surfactant types and quantities are used to make the membrane wettable, and not all surfactants are compatible with every recognition element to be striped.
    3. Elevated temperature vs. room temperature drying of the recognition element on the membrane result in differing arrangement and activity.
    Absorbant pad:
    1. Typically made of dense cellulose, must draw the sample continuously through the test strip.

    The main advantages of lateral flow assays is their ease of use. The assays are simple to perform and do not require specialized equipment or laboratory facilities. They are also relatively inexpensive compared to other diagnostic tests and can provide results within minutes. This makes them ideal for use in resource-limited settings where rapid and cost-effective diagnostic tests are needed.

    Custom Lateral Flow Assay Development

    If you are looking to develop your own lateral flow assays but find the process challenging, our assay development branch, CytoGroup, is here to assist. Specializing in the development of biosensors and diagnostic tools, CytoGroup offers comprehensive services to transform your concept into a fully functional commercial product. Our team of experts will guide you through every stage of development, from design to manufacturing, ensuring your assay meets the highest standards of quality and performance.

    Learn more about how CytoGroup can assist in your lateral flow assay development.

    Develop Your Own Lateral Flow Assay 

    For those aspiring to develop their own lateral flow dipsticks, Cytodiagnostics offers a wealth of resources to help you embark on this journey. We provide a vast array of research tools, materials, and kits tailored for lateral flow assay development. Our products are designed to streamline the development process, offering high-quality components and comprehensive technical support

    Whether you need specific reagents, specialized materials, or complete development kits, Cytodiagnostics equips you with everything required to start your LFA project. Our expert team is also available to offer guidance and insights, ensuring your path to successful assay development is smooth and efficient. Contact Us if you need help!

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