Key Area 1

(d) Detecting proteins using antibodies

Antibodies are roughly Y-shaped protein molecules (shown left) produced by lymphocytes. The two arms of the Y end in regions that vary between different antibody molecules (the variable region) - these regions are involved in antigen binding. The Y stem (constant region) is far less variable and interacts with effector cells and molecules.

Antibodies recognise and bind to specific antigens, found on the surface of pathogens.

Antibody-antigen complexes then clump together, making them easily targeted by other immune cells, including phagocytes.

Monoclonal antibodies are identical copies of one type of antibody, produced in a laboratory. While you do not need to know the details of how monoclonal antibodies are produced, you might find this short video (available at the top of this linked page) helps to solidify your understanding of this area.

Monoclonal Antibodies

Scientists can use immunoassay techniques to detect and identify specific proteins. These techniques use stocks of monoclonal antibodies that have the same specificity. An antibody specific to the protein antigen is linked to a chemical "label". A label is often a reporter enzyme producing a colour change.

Immunoassay - Pregnany test example

The image (right) shows an antibody (Y-shaped) bound to a specific antigen (green circle). The antibody is linked to a label (yellow starburst).

An ELISA (Enzyme-linked Immunosorbent Assay) is an example of an Immunoassay (depicted right). This might be used in diagnostic labs within hospitals - consider a patient concerned about prostate cancer. For people with prostate cancer, particular proteins are elevated, including a protein called PSA. A sample may be taken from the patient and added to an assay plate. A primary antibody with variable regions specific for PSA are added and bind to any PSA on the plate. After washing the plate to remove unbound material, a "secondary antibody" linked to a reporter enzyme is added to the plate, which binds, via its constant region, to any bound primary antibody. After another wash to remove unbound material, the substrate for the enzyme is added.

ELISA - Example of an immunoassay

The substrate will react with the enzyme on the secondary antibody, producing a coloured product. The concentration of antigen (e.g. PSA) is proportional to the colour intensity. This can therefore be used as a diagnostic tool for the detection of prostate cancer.

Immunoassays might use other chemical labels, rather than enzymes, including chemiluminescence, fluorescence and other reporters. In some cases, the assay uses a specific antigen to detect the presence of antibodies.

Fluorescent-labelled antibodies can be used in protein blotting and immunohistochemical staining of biopsied tissues (see right).

For more information, there is a tutorial available via the pink button below. Scroll down the website until you see "Tutorial".

This immunohistochemical image shows ovarian tissue biopsies stained using reporter antibodies specific for proteins known to be elevated in ovarian cancer. The brown areas show the presence of this protein.

Immunohistochemistry tutorial

Task 11

Testing for the Coronavirus, Covid-19, initially involved taking samples from a patient, sending it to the lab for PCR and then performing genetic sequence analysis. This is a very accurate but fairly time-consuming way to detect the presence of the virus. Alternative quicker immunoassay tests were soon developed. Can you suggest how these might work?

Answers are available here.

Western blotting

Western blotting is an analytical technique, used after SDS-PAGE electrophoresis, to detect and locate specific proteins from tissue extract. The separated proteins from the gel are transferred (blotted) onto a solid medium. The proteins can be identified using specific antibodies that have reporter enzymes attached. This process is shown in the diagram below.

Task 12

Read the Nature Education article by clicking on the pink box. Your task is to use this information to put together a short video or powerpoint to show how Western Blotting works.

Western Blotting Article - Nature Education