Antibody glossary

A collection of definitions from throughout the guide.

Adjuvant

Compounds/chemicals that are sometimes added to immunizing peptides during antibody production to help stimulate the immune response.

Affinity

A measure of the binding strength between antibody and antigen at a single binding site. Affinity refers to the strength with which the epitope binds to an individual paratope (antigen-binding site) on the antibody.

Affinity purification

Purification of an antibody based upon the strength of that antibody's binding to the antigen with which it was made.

Affinity constant

A numerical value indicating the strength of binding between antibody and antigen. It is also known as association or equilibrium constant.

Antibody

An immunoglobulin protein capable of specific binding with an antigen that has induced an immune response. Antibodies are produced by B cells as a primary immune defense in response to the presence of antigen.

Antigen

Any substance foreign to the body that elicits a specific immune response.

Antigen retrieval

Methods that enable an antibody to access the target protein within the tissue or cells. Typically used after fixation (particularly the use of paraformaldehyde) or embedding techniques that may cause chemical changes (eg, cross-linking of amino acids within the epitope) that mask epitopes and restrict antigen-antibody binding.

Antiserum

Blood serum from an immunized host containing antibodies of interest (as well as other serum proteins and antibodies).

Ascites fluid

Fluid taken from the abdomen of a living host animal, which will contain unpurified monoclonal antibodies produced by hybridoma cells grown within the host.

Avidity

A measure of the total binding strength of the antibody-antigen complex.

BSA

Bovine serum albumin. Often used as a carrier protein in antibody production.

Capture antibody

An antibody coating onto an ELISA plate that binds an antigen from an applied sample/solution.

Carrier protein

A large, highly antigenic molecule conjugated with a small antigen, called hapten. Carrier proteins induce a more effective and specific immune response in an immunized animal when creating antibodies for commercial use (eg, BSA or KLH).

Chromogen

A chemical substrate used to detect enzyme-tagged antibodies, for example, DAB. The chemical substrate will change color in the presence of the enzyme.

Conjugated antibody

Conjugated antibodies are chemically bound to labels, such as fluorochromes or chromogens, to enable visual detection of the antibody. Chemical substrates can be directly conjugated to a primary antibody or bound to a secondary antibody.

Cross-reactivity

The binding of an antibody to similar epitopes on other antigens or proteins.

Denatured

This term refers to the conformational change in an antigen away from its native state. Denaturing your sample may help expose an epitope or destroy it.

Detection antibody

The primary antibody used in sandwich ELISA, which detects the immobilized antigen. This antibody can be directly conjugated or visualized following the application of a conjugated secondary.

Direct staining

An antigen is detected by a primary antibody directly conjugated to a label (ie, conjugated primary antibody), so no secondary antibody is required.

Epitope

The specific site on an antigen molecule where an antibody binds via its variable region. Note that an epitope is not the same as an immunogen, which includes the epitope but is often much larger.

Fluorochrome (fluorophore)

A chemical compound that emits fluorescent light within a measurable color spectrum following excitation in response to a specific wavelength of laser light or chemical interaction.

Hapten

A small molecule that only elicits a specific antibody response when directly coupled to a larger carrier protein.

Hybridoma

A cell line created following the fusion of antibody-producing B cells from the spleen with an immortalized tumor cell line. A purified hybridoma culture/cell line will secrete its own specific monoclonal antibody. Read more about the hybridoma method of antibody production in Chapter 2.

Host species

The animal species in which an antibody has been raised.

Immunogen

A peptide sequence, chemical, or other substance capable of inducing an immune response

Immunogenicity

The ability of an antigen to induce antibody production.

Immunoglobulin (Ig)

General term for a family of proteins that function as antibodies, which includes several subclasses.

Indirect staining

An antigen is detected by a conjugated secondary antibody that has been raised against the primary antibody's host species and binds to the primary antibody.

Isotype control

An antibody of the same immunoglobulin subclass and from the same species as the primary antibody. This antibody is not raised against anything specific and is used to confirm the primary antibody binding is specific and not a result of non-specific Fc receptor binding or other protein interactions.

KLH

Keyhole limpet hemocyanin. Often used as a carrier protein in antibody production.

Monoclonal antibody

A homogenous population of antibodies that recognize a single epitope per antigen. Monoclonal antibodies are often produced using the hybridoma method.

Negative control sample

Any tissue, cell line, lysate, or purified protein known not to express/contain the antigen of interest.

Normal serum

Blood serum from non-immunized animals, which is often used as a control.

Paratrope

An antigen-binding site, ie, the antibody part that recognizes and binds to an antigen.

Optimal working dilution

Antibody concentration (or dilution) maximizing the positive signal while minimizing background and non-specific staining. Optimal working dilution must be optimized for different antibodies using a titration experiment.

Peptide

A short chain of amino acids.

Polyclonal antibody

A heterogeneous mixture of antibodies, with each antibody recognizing different epitopes of a particular antigen.

Positive control sample

Any tissue, cell line, lysate, or purified protein known to express/contain the antigen of interest.

Pre-adsorbed

An antibody is adsorbed with other proteins or serum from various species to eliminate any antibodies that may cross-react with a target of interest.

Pre-immune system

Serum extracted before immunization, which is often used as a control.

Primary antibody

The antibody that directly binds the antigen of interest. For direct staining, it will already be conjugated to a label. For indirect staining, a conjugated secondary antibody will be required for primary antibody detection.

Protein A/G purification

Column purification where the antibody's Fc domain binds the high-affinity S. aureus protein A or G.

RabMAb®

Abcam's patented technology for the generation of high-quality rabbit monoclonal antibodies.

Recombinant antibody

An antibody generated in vitro using synthetic genes.

Secondary antibody

A conjugated antibody that binds to the primary antibody and allows its visualization.

Specificity

This term refers to the antibody's ability to bind only the desired protein of interest. Various techniques can assess antibody specificity, including knock-out validation models.

Titration

An experiment designed to find the optimal antibody concentration for the desired application. The antibody is tested at a range of dilutions, and the results are assessed to identify the optimum concentration.