Selection of Secondary Antibodies
Determine in which species the primary antibody has been raised
Determine what subclass the primary antibody belongs to
Select a suitable marker for the secondary antibody
Make sure the secondary antibody works under the specific application condition
antibodies-online.com offers a broad range of various secondary antibody
anti-Immunoglobulin G (IgG) antibody
Secondary antibodies are usually designed to work in specific applications. For any given application several similar Secondary antibodies may be available. While primary antibodies are raised to detect a certain antigen secondary antibodies are designed to detect a primary antibody.
In order to do so, they are usually labeled with markers, or conjugates in order to detect and visualize the primary antibodies, most often via colorimetric detection or emission of a light signal. Secondary antibodies can be either
enzyme labeled (peroxidase, alkaline phosphatase), fluorescence labeled (e.g. FITC, Alexa-Fluor, Qdot) or biotin conjugated. Peroxidase is economical, rapid and a more stable enzyme, while the alkaline phosphatase on the other hand is considered more sensitive than peroxidase particularly when colorimetric detection is used. Fluorescent labeled antibodies are often used for double or multiple staining methods. Generally, any one of a range of markers or labels can be chosen, depending on the application and purpose of the investigation. In case of parallel detection of different antigens in the same sample (like on an IHC sample) one needs to select different monoclonal antibody classes (in order to avoid interference of secondary binding) and different markers in order to distinguish the antigen signals.
When choosing a secondary antibody, first of all the suitable secondary antibody for recognition of the correct primary antibody like IgG, IgM, IgE, IgD or IgA needs to be selected.
The significant difference between these isoforms are their heavy chains. They are denominated with Greek letters which are the basis for the antibodies' names: gamma, mu, epsilon, delta and alpha. Each antibody has two light chains (in mammals kappa or lambda) which also must be taken in to account for some applications (obviously if the secondary antibody is to bind not the Fc part or other regions of the heavy chains).
For the selection of the correct secondary antibody the specific subtype must be observed in most cases, i.e. if the subclass is IgG1, IgG2a, IgG2b, IgG2c, IgG3 and so on. However, general anti-IgG secondary antibodies may recognize various subtypes. Additionally, secondary antibodies can bind all parts of an IgG molecule (heavy and light chains, H+L), or only the Fab or Fc region, or only the gamma chain. Still others are specific for IgM heavy chains, or the lambda or kappa light chains common to all immunoglobulins (IgG, IgA, IgD, IgE, and IgM).
The secondary antibody will usually belong to the classes IgG or IgM.
A secondary antibody can further be defined as an antibody which binds to primary antibodies or antibody fragments. Secondary antibodies may be polyclonal or monoclonal, even though they most often derive from polyclonal sources and are affinity purified against the epitope they have been developed for. Secondary antibodies may hence be specific for whole Ig molecules or fragments of primary antibodies, such as the Fc or Fab regions.
Fc and Fab fragments can be generated with the enzyme papain that can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment. Proteolytic enzyme pepsin cleaves below the hinge region, forming a F(ab')2- and a pFc' fragment. The F(ab')2 fragment can be split into two Fab' fragments by reduction reaction.
For certain applications, antibody fragments are used as the primary antibody in order to obtain the best results. In that case a secondary antibody is used that is specific to the primary antibody fragment. For example, in
immunohistochemistry (IHC), background noise may be reduced if a Fab fragment of the primary antibody is used instead of the whole antibody molecule, because binding to endogenous Fc molecules in the sample does not occur with Fab-specific secondary antibodies.
Another situation occurs, when the secondary antibody itself is a fragment. For example, if working with tissues or cells that have Fc receptors (thymus, spleen, blood, hematopoietic cells, leukocytes, B cells, etc.) a F(ab')2 fragment secondary antibodies can be selected in order to avoid Fc receptor binding of the secondary antibody on the cells.
Host Species and Specificities of Secondary Antibodies
Most primary antibodies are produced in mouse or rabbit host species. Hence, anti-mouse IgG and anti-rabbit IgG are the most popular classes of secondary antibodies. However, there is a whole range of other specific antibodies. While monoclonal secondary antibodies are derived from mouse and rabbit, using the same basic techniques as for primary antibody production. Polyclonal antibodies for secondary antibody production are being produced in species such as rabbit, goat, sheep or donkey. Goat for instance is the host species most easily and frequently used by manufacturers to produce polyclonal anti-mouse and anti-rabbit secondary antibodies.
antibodies-online.com offers a wide variety rather common and very unusual secondary antibodies. Several kinds of anti-mouse and anti-rabbit secondary antibodies from other host species are also available. See the table below for help locating secondary antibodies with common or special host-species combinations and various conjugation options.