Functioning of a lateral flow test

Immunochromatographic rapid tests (also termed Lateral Flow Assays) were originally developed in the late 1980s as a rapid and simple pregnancy test. Since then, this method, which neither needs trained personnel nor dedicated laboratory equipment, has gained wide acceptance for a variety of point-of-care and field-use applications in human, veterinary and consumer diagnostics. The following paragraph briefly discusses the main components of a Lateral Flow Assays and gives an overview of the employed assay formats.


The drawing shown above depicts the typical setup of a lateral flow assay. It consists of a sample- and conjugate pad, a nitrocellulose membrane and a wicking pad. The single components overlap each other and are fixed on a plastic backing material.


Samples are first applied to the sample pad. Its function is to adjust the sample for the following components of the test. Special whole blood separators might be used as sample pad to ensure that only serum reaches the second component of the test, namely the conjugate pad. A particulate conjugate has been laid down on the conjugate pad. The particles are mostly made of gold but latex may likely be used. The particles are linked to the first biological component of the assay (an antibody or antigen) and as the sample reaches the conjugate pad, it rehydrates the conjugate and thus allows a reaction between the analyte and the biological component linked to the gold particle. This complex then migrates onto the third component of the assay: the reaction matrix. The reaction matrix typically consists of a nitrocellulose membrane to which the other specific biological components of the assay have been applied. The test line typically consists of proteins (antigens or antibodies) which have the function to capture the primary analyte-antibody complex and ensure the generation of a color signal. Conjugated particles that have not bound to the test-line bind to the control line, where in most cases a species-specific immunoglobulin, which reacts specifically with the antibody conjugated to the colloidal gold, is immobilized. Thus a color signal is generated at the control line irrespective of the result at the test line. The fourth component of the lateral flow assay is the wicking pad. It adsorbs the fluid from the nitrocellulose membrane and acts as the motor of the whole assay. The sample formats used in lateral flow assays are either direct (sandwich immunoassay) or competitive (inhibition immunoassay) and allow qualitative, semi-quantitative in some selected cases quantitative determinations of the analyte.


These direct rapid test systems are used when larger analytes with multiple antigenic determinants, such as  e.g. a parvovirus detection of our Fassisi ParCo, is to be detected. In this case, a positive result is indicated by the appearance of a test line and a control line.


The following movie demonstrates the mode of operation of such a sandwich immunoassay. As an analyte is present in the sample both the test and control line appear in the assay.