Lateral-flow testLateral flow tests are also called immunochromatographic strip (ICS) tests or simply strip-tests. They have been a popular platform for diagnostic tests since their introduction in the late 1980s.

Lateral flow tests are used for the specific qualitative or semi-quantitative detection of many analytes including antigens, antibodies, and even the products of nucleic acid amplification tests. One or several analytes can be tested for simultaneously on the same strip. When used as a clinical diagnostic urine, saliva, serum, plasma, whole blood, feces, exudates (from wounds or lesions) can all be used as specimens.  In environmental or other non-clinical applications the sample may be derived from soils, dust, vegetation, or food, or environmental swabs such as from food processing plants.

Lateral flow tests are the simplest to use of all of the test formats described on this website, simply requiring the user to place the test strip in the specimen or added directly onto the strip itself and read the results after a specified amount of time.  Results can usually be read in as little as 2 minutes, but around 15 minutes is more common. Many lateral flow tests have limitations on how long one should wait before reading them, thus if you wait to long to read the results the assay will need to be repeated.  All tests include either a procedural control line or a sample adequacy control line that is used to validate the test result. Appearance of two lines, therefore, indicates a positive result, while a valid negative test produces only the control line. If only the Test line appears, or if no lines appear, it is invalid and must be repeated.

The basic components of the lateral flow/ICS test

In one of its more common forms the Lateral Flow strip is composed of roughly 7-9 components. These are:

Basic components
Click for details.
  1. Sample Pad
  2. Conjugate Pad- The conjugate pad contains detection particles (conjugate) adsorbed with antibodies or antigens specific to the analyte being detected
  3. Detection Conjugate
  4. Solid-phase Membrane
  5. Test and control reagent lines
  6. Absorbent Pad
  7. Plastic-adhesive backing card

The following components are “optional” and are not necessary or included in many lateral flow platforms.

  1. Laminate Tape
  2. A Strip housing/Cassette

For the most part, all the components are layered on to the plastic backing card. Each component must be carefully positioned so that they overlap. This allows the reagents and sample and buffers flow up through the membrane and to the adsorbent pad.

The assembled strips are dried and packaged, making them stable for months when properly protected from moisture and excessive heat.

Summary of how a lateral flow test works:

Step 1
Click for details.

To perform the test, a sample is placed on the sample pad at one end of the strip. The sample may be used alone as is commonly done with urine or serum compatible tests, or it may be mixed with a buffer specific to the test. This buffer may simply be a diluent/running buffer or it may be much more complex and have specific components or properties required to make the strip perform properly, such as a cell lyses buffer. In the following description we are assuming a gold conjugate is being used. While this is one of the most common detection methods; however it is certainly not the only one available.

Step 1
Click for details.

With the addition of the sample, the detector molecules are solubilized. When solubilized the detector molecules mix with and bind to the analyte in the sample (if analyte is present).

Step 3
Click for details.

Then capillary action draws the fluid mixture up the sample pad and into the membrane. The sample/detector molecule mix continues to move up the membrane until it reaches the analyte capture molecule. In these lines a second (and third) antibody or antigen, immobilized as a thin stripe in the nitrocellulose will then capture the complex if it is positive for the target analyte. The control line should always show as a visible line, otherwise the test is invalid and must be repeated. If the test is positive, a colored (typically pink or purple) line develops along with the control line.

Step 4
Click for details.

Excess buffer along with any reagents not captured at the test of control line will then move into the absorbent wicking pad.