Lateral Flow Immunoassay

Creative Diagnostics - Food & Feed Analysis

What Is A Lateral Flow Immunoassay?

Lateral flow test strips based on the principles of immunochromatography exist for a wide array of target analytes. The fist tests were made for the detection of human chorionic gonadotropin (hCG). Today, there are commercially available tests for ovulation monitoring, infectious disease detection, drugs of abuse analysis, and measuring other analytes important to human physiology. Products have also been introduced for testing, agricultural applications, environmental testing, and food & feed testing.
While the fist 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.
A lateral flow immunoassay is described using varying terminology by different sectors and different countries. Common names include:
+ Lateral flow immunoassay (LFIA)
+ Lateral flow test (LFT)
+ Lateral flow device (LFD)
+ Lateral flow assay (LFA)
+ Lateral flow immunochromatographic assays
+ Dipstick
+ Rapid test
+ Test strip
+ Quick test

Types of Lateral Flow Tests

Lateral flow assays can be developed to be used in a dipstick format or in a cassette. Both dipsticks and cassette tests will work in a similar way, it is just dependent on the industry, sample matrix, and the market requirement, as to which format is suitable.

Sandwich Format

The sandwich assay format is typically used for detecting relatively large analytes. If the analyte has at least two distinct binding sites (i.e. epitopes), a “sandwich” assay can be developed where an antibody to one epitope is conjugated to the nanoparticle and an antibody to another epitope is immobilized at the test line. The sandwich format results in a signal intensity that is proportional to the amount of analyte present in the sample.

Competitive Format

A competitive format is used for detecting analytes in which the analyte is too small for two antibodies to bind simultaneously, such as vitamins and antibiotics. In a competitive assay, the test line contains the target analyte molecule (usually a protein-analyte complex). The nanoparticles are conjugated to an antibody that recognizes the analyte. If the analyte is not present in the sample, the nanoparticle antibody conjugates will bind to the analyte at the test line, resulting in high signal intensity. If the target analyte is present in the sample, the analyte will bind to the antibodies on the nanoparticle surface and prevent the nanoparticle from binding to the test line. This will reduce the signal at the test line resulting in a signal intensity that is inversely proportional to the amount of analyte present in the sample.

Traditional Lateral Flow Components

The lateral flow immunoassay technology use nitrocellulose membrane, colored nanoparticles (or labels), and typically antibodies, to produce results.
When a sample is added, the sample will flow along the test device passing through the conjugate pad into the nitrocellulose membrane and then onto the absorbent pad.

The figure below demonstrates how a sandwich assay works:

Sample pad: Acts as the first stage of the absorption process, and in some cases contains a filter, to ensure the accurate and controlled flow of the sample.
Conjugate pad: Stores the conjugated labels and antibodies, will receive the sample. aids in controlled release of re-solubilized conjugate onto the nitrocellulose membrane.
Nitrocellulose membrane: Provides the ideal solid phase for immobilizing test and control line reagents. As the sample moves along the device the binding reagents situated on the nitrocellulose membrane will bind to the target at the test line. A colored line will form and the density of the line will vary depending on the quantity of the target present.
Absorbent pad: Provides uniform capillary flow through the membrane, absorbs applied sample, and prevents backflow.

Sample Matrices

The target analyte and the market requirements will determine the type of sample that will be used in the assay.
Some samples require running buffer to aid sample delivery e.g. animal feed. Other samples such as blood, serum, urine, or saliva may be able to be placed directly onto a test, while there are occasions where a dilution buffer is required.
Lateral flow immunoassays are developed to detect target analytes in sample matrices including:
1. Milk
2. Whole blood
3. Serum
4. Saliva
5. Urine
6. Tissue samples
7. Food
8. Drink
9. Animal feed
10. Plant material
11. Water

Label Types

Typically, lateral flow assays utilize conjugated gold nanoparticles within the conjugate pad. Other labels include colored polystyrene beads, magnetic beads, quantum dots or upconverting nanoparticles.
The optimization of the assay will ensure the label interacts correctly with the antibody and antigen to ensure efficiency and accuracy of results.

Multiplexed Lateral Flow Assays

Both sandwich and competitive assays can be developed to include one or more test lines.
A multiplexed assay may be used for:
Detecting multiple targets in a single test rather than using many individual tests. In situations where only a small sample volume is available a multiplex assay allows you to maximize its use;
To assist diagnosis where the presence of a number of markers together is required;
Confirming the presence of multiple contaminants during high volume food and feed testing;
Offering cost-saving benefits to end-users in a laboratory or in-the-field by testing for different targets simultaneously;
Remote or agricultural areas where resources are limited and where multiplexed testing will save time.

Quantitative Rapid Lateral Flow Devices

Early versions of LFDs were predominantly qualitative assays. However, improvements in reagents, component materials, and reader technologies along with manufacturing processes mean quantitative results are achievable.
In addition, the developments in reader technology and advancements in raw materials, such as labels, means a lateral flow rapid test can match the sensitivity of an ELISA assay.

Advantages & Disadvantages of Lateral Flow Immunoassays

Advantages Disadvantages
Low cost Qualitative or semi-quantitative readout
Wide range of applications Restriction on total test volume can impose a limit on sensitivity
Well-established technology, ease of manufacture Inaccurate sample volume may reduce precision
Long shelf-life, no need for refrigeration Test-to-test reproducibility may be problematic
Simple, user-friendly operation Difficult to miniaturize sample volume
High sensitivity and specificity Multiplexing can be challenging
Low sample volume required Unclear patent situation in some instances
One-step assay, no washing steps necessary, short time to result
Relatively short timeline for development, time to market is reduced
High potential for commercialization
Easily scalable
Can be integrated with reader systems
Possibility of multiplexing
  1. Advantages :
    Low cost
    Wide range of applications
    Well-established technology, ease of manufacture
    Long shelf-life, no need for refrigeration
    Simple, user-friendly operation
    High sensitivity and specificity
    Low sample volume required
    One-step assay, no washing steps necessary, short time to result
    Relatively short timeline for development, time to market is reduced
    High potential for commercialization
    Easily scalable
    Can be integrated with reader systems
    Possibility of multiplexing
  2. Disadvantages :
    Qualitative or semi-quantitative readout
    Restriction on total test volume can impose a limit on sensitivity
    Inaccurate sample volume may reduce precision
    Test-to-test reproducibility may be problematic
    Difficult to miniaturize sample volume
    Multiplexing can be challenging
    Unclear patent situation in some instances

If you would like to speak with us about our lateral flow immunoassay development services, please contact us on

Contact Us
  • USA
  • 45-1 Ramsey Road, Shirley, NY 11967, USA
  • 1-631-624-4882
  • 1-631-938-8221
  • Europe
  • 7 Bell Yard, London WC2A 2JR, UK
  • 44-161-818-6441
  • Creative Diagnostics
Inquiry Basket