Lab 8 Background

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is a technique used to detect antigens in biological samples by taking advantage of the specific binding between antigens and their corresponding antibodies.

An antigen is a molecule that belongs to a foreign object that can be recognized and bind to a specific antibody or a T cell receptor, which then may trigger an immune response. An antibody is a Y-shaped protein complex made by the immune cells to identify and neutralize foreign objects. Each antibody recognizes and binds to a unique antigen.

In an ELISA assay, an antibody that can recognize a specific antigen is used to detect the object that the antigen belongs to. In our example, the antigen comes from part of a unique T7 phage protein. Hence, the antibody, we can call the T7 antibody will only recognize the T7 phage and not detect non-T7 phage objects in the sample.

Because the T7 phage-T7 antibody binding is not visible to the human eye, a method is needed to visualize the binding. This is done through the binding of a secondary antibody that is labelled with a chemiluminescent molecule. The secondary antibody is specially made so it can bind to the primary antibody, and because the primary antibody binds to the target of interest, the secondary antibody is also attached to the target. The use of a chemiluminescent molecule is important because once activated, the molecule can produce a color that signals the detection of the target; the intensity of the color also reflects the relative amount of the target.

In the lab, the primary T7 Phage antibody will be pre-applied to filter papers, and you will add a set of samples with known concentrations and your phage sample to the filter papers to allow antigen-antibody interaction. After washing off the extra non-binding molecules in the samples, a secondary antibody will then be applied to the filtered papers. After washing, a reagent will be added to allow the chemiluminescent molecule to generate color on the filter papers. The papers will then be scanned and the relative color intensity of the filter papers will be analyzed using simple software. The intensity should correlate with the viral titer of the samples.

Reference