Lateral Flow Tests - What are they?

A Lateral Flow Test, also called LFT, immunochemical test strip, immunochromatigraphic test, dipstick test, to detail a few names, is a simple diagnostic test used to detect the presence or estimate the concentration of a specific substance in a liquid sample. This can be a protein, virus, bacteria, drug, etc. which can be detected from a nasal fluid, saliva, blood, serum, plasma, feces or urine sample. These tests are rapid, near-patient tests that usually can yield a result in around 10-15 minutes without requiring complex laboratory equipment. Common examples include pregnancy tests, COVID-19 rapid antigen tests, and rapid tests for certain infectious diseases (Flu, RSV, Lymes, etc.) They can be developed in 2 formats: a competitive Inhibition binding assay (as shown in the diagram above) or a non-competitive Sandwich LF assay (as shown in the diagram above).

How are Lateral Flow Tests made?

Lateral flow tests usually use antigen–antibody interactions to detect a target substance. The liquid sample moves along a porous membrane by capillary action, carrying the sample and detection reagents that will produce a result when the target is present.
A typical lateral flow assay consists of five main components assembled on a mylar backing card.

Sample Pad

• Material: Cellulose, glass fiber, or another porous material.
• Function: Receives the liquid sample and distributes it evenly across the strip.
• It may be treated with buffers, surfactants, or other chemicals to adjust the sample's pH, reduce unwanted interactions, and improve flow.

Label Pad

• Material: Usually glass fiber or another porous material that releases reagents when wetted.
• Function: Stores the labeled detection reagent in a dry, stable form.
• When a sample is added to the test, the labeled reagent in this pad dissolves and moves with the liquid.
• The label entity, can be colloidal gold, colored microspheres, or fluorescent molecules.

Membrane

• Material: Usually nitrocellulose, although other membranes may be used.
• Function: the main reaction support for the test. It minimally contains one test line and one control line.>

  Test line

  • Contains immobilized capture molecules, often antibodies.
  • Captures the target or a target–detection reagent complex.
  • Produces a result if sufficient labeled material accumulates at the line.

  Control line

  • Contains immobilized molecules that capture the labeled detection reagent, typically regardless of whether the target is present.
  • Confirms that the liquid has flowed through the strip and that the detection reagent has functioned sufficiently for the control reaction.

Absorbent pad

• Material: Highly absorbent cellulose or similar material
• Function: Draws liquid through the membrane and collects excess sample.
• Maintains capillary flow along the membrane.

Backing card and housing

• Backing card: The support upon which the membrane and pads are aligned. The pads and membrane are arranged with slight overlaps so that liquid can move continuously from one component to the next.
• The plastic housing protects the strip, helps users apply the sample correctly It provides a window through which the result can be read.
• Maintains the capillary flow along the membrane.

How do Lateral Flow Assays work?

The working principle is based on capillary action, specific molecular recognition, and the accumulation of labeled particles. A liquid sample is added to the sample pad. The sample moves through the sample and reaches the label pad by capillary action and dissolves the dried detection reagent. The liquid carries the labeled reagents and any resulting complexes along the membrane. At the test line, immobilized capture antibodies recognize the target or another part of the complex. If a non-competitive assay format is used, the labeled particles accumulate at the test line, producing a colored band. If a competitive assay format is used, the labeled particles will not accumulate at the test line. In either format, unbound detector reagent will continue up the membrane and will be captured at the control line. The control line indicates that capillary flow along the membrane proceeded as expected. It does not guarantee that the test has worked perfectly.

There are two major formats used for antigen or antibody detection: A non-competitive format (refered to as a Sandwich lateral flow assay) and a competitive format. The non-competitive format is commonly used when the target is large enough to bind two recognition molecules simultaneously. A positive result produces a visible signal.

Examples: are Pregnancy tests and covid tests. Both have large target molecules

The competitive lateral flow assayformat is often used for small molecules that cannot bind two antibodies simultaneously because they have only one suitable recognition site. The target in the sample competes with a target-like substance or immobilized target reagent for binding to the detection reagent.

Examples include drugs, drugs of abuse, steroids, and other small molecules.

An important difference between the 2 formats: In a non-competitive assay, a test line appears and gets stronger for a positive result, while in a competitive assay, a test line gets weaker or even disappears for a postivie result.

How to run a Lateral Flow Assay

1. You apply a sample (dissolved in a liquid, if necessary) to the dipstick or the sample well of a test housing.
2. You start a timer and set it to the prescribed time in the test's Instructions for Use.
3. You read the result at the prescribed time.
4. The test line can yield a visual signal, or signal disappearance, discernable by eye, or can yield a fluorescent signal, discernable with an intrument.
5. A second line (control line) will appear if the liquid applied to the membrane flowed correctly.