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Lateral Flow Immunoassay (LFIA EXPLAINED)

Before we dive into the immunoassay part of the test, we first should understand the working principle behind lateral flow or LF tests. My bachelor’s thesis actually discussed the use of lateral flow tests in great detain so this is a topic near to my heart! All LF tests consist of three main pads plus a membrane. These different parts in order are the sample pad, the conjugate pad, the membrane and absorbent pad. The conjugate pad also contains one or several test lines, where the target analyte can bind, in addition to a control line which ensures the functionality of the test. Let us take a look at what happens when we apply a sample to a lateral flow immunoassay: 1. First, the sample gets added to the *sample pad* which promotes and distributes the sample evenly towards the conjugate pad. 2. The *conjugate pad* is where the test line is located. In the case of lateral flow immunoassays, this is usually a *labeled* *monoclonal antibody* which has been dried directly on the pad. Once the sample reaches this pad, if the target analyte, i.e. the sought after substance is present it binds to the monoclonal antibody. 3. Then the sample molecules, some of which have bound to the antibody flows to the **membrane**, which is usually made of nitrocellulose. It is essential that the membrane possesses good capillary forces and ease of binding. Here is where both the test line and control line is located. The test line is the first of these two and is coated with monoclonal antibodies that are also specific to the antigen of the target analyte. This means that if we can detect a signal at the site of the test line, the target analyte has successfully bonded twice to two different monoclonal antibodies specific to it. Then the unbound labeled antibodies continue to the control line where they bind to a secondary antibody. Thus, a line from their label forms here as well and this allows us to check that the test is not faulty! 4. Finally the sample flows through to the **absorbent pad**. The primary function of the absorbent pad is to wick the fluid through the membrane. Cellulose filters are commonly used as the material for these. When the absorbent pad is present, the amount of sample can be increased, improving the sensitivity of the test. So why, are these so useful? Well, it is more than likely that you have encountered one of these lateral flow immunoassays at least once in your life, and probably more these last few years. LFAs are commonly used for home pregnancy tests as well as the COVID tests.