Different types of lateral flow assays
In a small, paper-based system, lateral flow assays detect a compound of interest. Pregnancy tests, for example, are based on lateral flow assays, and there are many more available for various compounds or even pathogens. Samples are treated in such a way that compounds of interest bind to an antibody-conjugate, allowing them to be visualized.
Two lines, known as the test and control lines, are present within the detection zone; the test line indicates the presence of a compound of interest, and the control line indicates that the sample has passed the test line. The type of lateral flow assay will be described in detail based on how the compound of interest is detected.
Lateral flow immunoassays
Antibodies are used in lateral flow immunoassays to detect the compound of interest. There are two types of lateral flow immunoassays:
Sandwich lateral flow immunoassay
Sandwich lateral flow immunoassays are used in HIV testing and pregnancy tests. On the test line, antibodies that detect the compound of interest are placed. Then, a different antibody that also binds to the compound of interest is labeled and mixed with the sample to form an antibody-conjugate.
When the test is performed, the compound of interest will bind to both the antibody-conjugate and the antibodies on the test line. As a result of the antibody-conjugate binding to the compound of interest, the test line becomes visible.
Immunoassay for competitive lateral flow
There are two more divisions in the competitive format of lateral flow immunoassays.
The compound of interest is immobilized onto the test line in the first division. Antibody-conjugates are mixed with the sample as usual, but the methodology of the two types of competitive immunoassays diverges after this first step.
In this first format, the antibody conjugate binds to the test line only if it has not bound to the compound of interest within the sample prior to reaching the test line; that is, if the sample contains no compound of interest, the antibody-conjugate is free to bind to the test line; however, if the sample contains a high concentration of the compound of interest, the antibody-conjugate will bind the compound in the sample rather than the test line.
The compound of interest is immobilized on the test line in the second division, this time by binding to the antibodies on the test line. This means that if the antibody-conjugate contains the compound of interest, it cannot bind to the test line because the compound of interest is already present in the test antibodies. If no compound of interest is present in the sample, the antibody-conjugate binds to the compound immobilized on the test line.
This means that a positive result will not produce a visible line on the test line, whereas a negative result will produce a visible line on the test line for both divisions.
What is the advantage of using a competitive lateral flow immunoassay? - Some small molecules have only one "antigenic determinant" capable of binding antibodies. As a result, using two antibodies as in the sandwich format is not possible.
Advances in lateral flow assays
In recent years, advances in lateral flow assays have resulted from new ways to enhance signals, new labels, improvements in quantification, and the detection of multiple compounds of interest at the same time. Silver enhancement technology, for example, has been used to improve the detection signal of gold nanoparticles.
Another method involved using enzymes found within gold nanoparticles to create a different type of antibody-conjugate. These enzymes, such as horseradish peroxidase, can degrade (catalyze) compounds to form a colored substrate, which can then be detected on the test line and may be more sensitive than traditional antibody-conjugates alone.
