Assay Resource Guide

Immunoassays are highly utilized techniques that are performed in research laboratories worldwide. Use our assay resource guide to learn about the various types of immunoassays available.

Overview of immunoassay techniques

An immunoassay, also known as immunodetection, immunostaining, immunolocalization, or immunolabeling, describes a broad-range of techniques that use an antibody to detect a specific target protein in a biological sample.

Immunoassay tests include:

Western Blotting (WB)

Immunofluorescence (IF)

Immunocytochemistry (ICC)

Immunohistochemistry (IHC)

Immunoprecipitation (IP)

Enzyme-linked immunosorbent assay (ELISA)

Flow Cytometry (FCM)

Immunoassay Labels

Immunoassays use a variety of labels (also known as stains, tags, dyes, or conjugates) to visualize the detected antigen. These include enzymes (horseradish peroxidase, alkaline phosphatase), fluorophores (fluorescein, rhodamine, phycoerythrin, ATTO dyes) or electrochemiluminescent tags. Immunoassays are often grouped into subcategories based on the antigen detection system used.

Direct vs Indirect Immunoassays

Immunoassays can be conducted using either direct or indirect labeling.

Direct immunoassays are conducted using an antibody that is directly conjugated to a label (fluorophore or enzyme). This conjugated antibody will then bind the antigen, allowing for direct visualization of the target protein.

Indirect immunoassays are conducted when the antigen is detected by a primary antibody that is unlabeled. Then a secondary antibody that is conjugated to a label (fluorophore or enzyme) is added to detect the primary antibody.

What is a Western blot?

Western blotting (also known as a protein immunoblot) is used to determine the molecular weight of an antigen present in a complex mixture of tissue or cell lysate. Proteins are first separated by size using gel electrophoresis then transferred to a membrane (PVDF or nitrocellulose) and stained with target specific antibodies.

Assay sensitivity can be modulated by using different enzyme substrates. The standard colorimetric western blot will likely use an antibody-peroxidase conjugate. If a higher sensitivity is required, the antibody-peroxidase conjugate is replaced with a biotin-streptavidin conjugate.

Enhanced chemiluminescence western blotting (ECL) involves conjugating HRP to the target antibody, which will cause a catalytic conversion to produce an even higher signal, allowing the detection of minute quantities.

 

What is Immunofluorescence?

Immunofluorescence (IF) Definition: Detection of a target using an antibody and subsequent visualization using a fluorophore

Immunohistofluorescence (IHF) Definition: Detection of a target in tissue using an antibody and subsequent visualization using a fluorophore.

Immunocytofluorescence (ICF) Definition: Detection of a target in cells using an antibody and subsequent visualization using a fluorophore.

 

What is immunohistochemistry?

Immunohistochemistry (IHC) Definition: Detection of a target in tissue using an antibody and subsequent visualization using a chemical reaction to produce a color change.

Immunohistochemistry staining is used to identify target proteins in tissue sections (containing both cells and an intact extracellular matrix) using antibodies. Proper fixation is crucial to successful staining. Formaldehyde fixation is often used as the routine initial method of choice for tissue.

To detect a target protein, the primary antibody is often labeled directly with an enzyme. A substrate can then be added to react with the enzyme and create a colored product. Alternatively, the primary antibody signal can be amplified by conjugating it to many biotin molecules. Similarly, a biotinylated secondary antibody specific for the primary antibody can be added. Then upon addition of a streptavidin-enzyme conjugate, the streptavidin will bind strongly to the biotin molecules.

However if further amplification is desired, a biotin-enzyme conjugate can be incubated with streptavidin to form large avidin-biotin-enzyme complexes. The tissue can then be incubated with this complex, labeling each biotin molecule with numerous enzymes, as each streptavidin can bind up to 4 biotin molecules. Finally, the substrate is added causing a colored precipitate to form on the tissue at the location of the antigen. This tissue is then analyzed using a light microscope or embedded and prepared for electron microscopy.

 

What is immunocytochemistry?

Immunocytochemistry (ICC) Definition: Detection of a target in cells using an antibody and subsequent visualization using a chemical reaction to produce a color change.

Immunocytochemistry staining is used to identify a specific target protein in samples of intact cells without the surrounding extracellular matrix found in tissues.

 

What is immunoprecipitation?

Immunoprecipitation is used to isolate a protein of interest from the rest of a sample. It has a variety of applications including determining the molecular weights of protein antigens, to study protein-protein interactions, to determine specific enzymatic activity, to monitor protein post-translational modifications, and to determine the presence and quantity of proteins. Generally speaking, an antibody for the protein of interest in incubated with a cell extract so that the two bind in solution. The antibody-antigen complex will then be pulled out of the sample using protein A/G-coupled agarose beads. The sample can then be separated by SDS-Page for western blot analysis.

 

What is an ELISA?

Enzyme-linked immunosorbent assay (ELISA) is a diagnostic tool that uses an enzyme-immunoassay (EIA) to detect the presence of an antigen. A sample with an unknown amount of antigen is first immobilized on a plate. The addition of the secondary antibody forms an antibody-antigen complex. The secondary is usually conjugated to an enzyme which will produce a visible signal when the plate is developed. This signal will indicate the quantity of the antigen in the sample.

There are three main types of ELISAs, direct ELISA, sandwich (indirect) ELISA and competitive ELISA.

 

What is Flow Cytometry?

Flow cytometry is a method by which suspended immunolabeled cells are counted and analyzed based on their physical and chemical characteristics. It is used to differentiate cell types from a heterogeneous population based protein expression, cell size, and volume. Flow cytometers can analyze a few thousand cells every second, and output multi-parameter datasets.

Fluorescence-activated cell sorting (FACS) is a variation of flow cytometry that sorts a population of cells based on their fluorescent labels into separate containers so that they can undergo further analysis.