Lateral Flow Immunoassay

The backing material. This material is usually plastic in nature and features a pressure sensitive adhesive for the other material to stick to. It can be white (for normal applications) black for fluorescent particles and translucent for specialty applications like transmission based optical measurement. Adhesive choice is important for eventual high scale manufacturing.

Nitrocellulose. This material is made much like writing paper – and is the reason why a lot of people call lateral flow tests – paper-based tests. However, this material is highly specialized for pore size and protein binding. There are many options available and can be optimized for speed, sensitivity and precision. Liquid reagents like antibodies or small molecule-protein conjugates are sprayed down and dried in discrete lines on this material to form control and test lines – prior to lamination. Two major categories of lateral flow immunoassays exist. Sandwich and competitive. In a sandwich assay two antibodies are used (one on the nitrocellulose and one coupled to a colored particle) to capture and label the antigen (the target to which the antibody binds). Antigen concentration is directly proportional to the resulting color intensity on the test line. In a competitive assay – the antigen of interest is immobilized on the nitrocellulose and an antibody raised against that antigen is coupled to a colored particle. Antigen in the sample competes for binding sites on the antibody with the test line. Antigen concentration is inversely proportional to the resulting color intensity on the test line. Lastly, this material is the reason for the term “chromatographic” in “ICT (immunochromatographic Test). Nitrocellulose mimics the action of Thin layer Chromatography plates used in laboratories. It is this action that led the original lateral flow researchers (such as our founder Paul Davis) to the take technology further. The ability of nitrocellulose to separate free from bound is what makes lateral flow immunoassays work!

Absorbent / wicking pad. This material is usually cotton based, and its function is to increase capillary force of the sample liquid across the nitrocellulose membrane. These come in various lengths and grades. It’s an often-overlooked component – but can have dramatic effects on precision if the choice is not made correctly or if not enough length for the amount of sample volume added is used.

Sample / Conjugate pad. This material is usually made out of woven glass fibers, but can be with synthetic polymers as well. Sometimes, a second sample pad is used in addition to this pad (as in the case of a blood separation pad). When used as one or separately, the sample / conjugate pad can be pretreated and dried with buffers, salts, surfactants and other chemicals to facilitate proper addition of sample to the test strip. The goal for these materials is to accept the sample and then allow it to move on to the nitrocellulose once it has picked up the components dried down on the pad.

Conjugate / Label. This liquid is dispensed through something that resembles a mini spray paint nozzle using pressurized air. It is the second part of the reaction and contains either colored particles or enzymes that aggregate on the test and control line to visualize the result. Dry down and reconstitution buffers are highly specialized for the label being used. Particles include colloidal gold of various sizes, dye doped latex of various sizes, and Europium (fluorescent). Enzymes require the addition of precipitating dye substrates which can be incorporated with GADx designed cassette housings. The antibody is conjugated to these particles or enzymes prior to being dried down in very specialized processes. The selection of type and size of label is paramount to assay performance. Once dried onto the sample / conjugate pad – the antibody – particle conjugate is stable and can be easily reconstituted by the sample.