Overview of Secondary Antibodies

A secondary antibody aids in the detection, sorting or purification of target antigens by binding to a primary antibody, which directly binds to the target antigen. The use of secondary antibodies to indirectly detect target antigens requires more steps than using the primary antibody alone but can also offer significant advantages over primary antibodies that are directly labeled. Secondary antibodies offer increased sensitivity through the signal amplification that occurs as multiple secondary antibodies bind to a single primary antibody. In addition, a given secondary antibody can be used with any primary antibody of the same isotype and target species, making it a more versatile reagent than individually labeled primary antibodies.


Because the vast majority of primary antibodies are produced in just a few host animal species and most are of the IgG class, it is economical to produce and supply ready-to-use secondary antibodies for many methods and detection systems. From a relatively small number of secondary antibodies, many options are available for purity level, specificity and label type for a given application.


Specificity of Secondary Antibodies

Secondary antibodies are generated by immunizing a host animal with an antibody from a different species. For example, anti-mouse antibodies are raised by injecting specific purified mouse antibody into an animal other than a mouse. Goat, donkey, sheep, chicken and rabbit are the most commonly used host species for raising secondary antibodies, though others are available.


The most common types of secondary antibodies are those generated against a pooled population of immunoglobulins from a target species. For example, immunizing a goat with purified mouse IgG will generate goat anti-mouse IgG antibodies that will bind to all classes, heavy and light chains (H&L) and fragments of mouse IgG, as well as any other molecules sharing the same conserved domains (e.g., IgM share the same kappa light chains as IgG). In contrast, immunizing a goat with only mouse IgG1 antibodies will only generate antibodies specific for mouse IgG1 antibodies and molecules sharing the same conserved domains.


Because of the high degree of conservation in the structure of many immunoglobulin domains, class-specific secondary antibodies must be affinity purified and cross-adsorbed to achieve minimal cross-reaction with other immunoglobulins. Using the example described above, immobilized mouse IgG1 antibodies would be used to affinity purify all goat antibodies that bind to mouse IgG1. These anti-mouse IgG1 antibodies would then be further purified by passage through a chromatography column(s) containing mouse IgG2a, IgG2b, IgG3, IgM, etc., to remove any antibodies that cross-react with non-IgG1 isotypes.


Additionally, secondary antibodies can be further purified by passage through columns containing the immobilized serum proteins from species other than those used to immunize the host. This method of cross-absorption (referred to as "Cross-Adsorbed") is an additional purification step recommended for applications where primary antibodies from multiple species will be used and when immunoglobulins or other serum proteins may be present in the samples being probed.


Secondary Antibody Fragments

Secondary antibodies may be provided in several formats: whole IgG, divalent F(ab')2 fragments and monovalent Fab fragments.


Whole IgG

The most widely used secondary antibodies are whole IgG molecules. They are typically affinity purified from the pooled serum from a host immunized with the desired purified immunoglobulin from a different species. Isolated IgGs can then be further purified and depleted against unwanted cross-reactive proteins or immunoglobulins from other species to further enhance specificity.


F(ab')2 antibody fragments

While whole immunoglobulins are compatible with most assays, certain methods benefit from removing the Fc portion of the antibody in order to reduce the mass of the antibody, due to cross-reactivity of other antibodies with host Fc, or because the probed samples contain active Fc-binding proteins (e.g., Fc receptors, IgG-like domains, Protein A, Protein G). The Fc portion can be removed from several species of IgG by digestion with pepsin, leaving the divalent F(ab')2 fragment (~100 kDa) of the antibody intact.


Fab antibody fragments

Most species of IgG can be enzymatically digested with papain to cleave the antibody between the antigen binding domain and the hinge region to produce two Fab fragments and an Fc fragment. The monovalent Fab antibody fragments are useful in blocking applications and other special circumstances where controlled binding ratios and/or the elimination of Fc interactions is required. The small size (~50 kDa) of Fab fragments may improve antigen detection by penetrating deeper than whole IgG into tissue sections and other complex samples. Fab fragments can also be useful in targeting intracellular antigens as they are small enough to pass through the membrane in living cells.


Secondary Antibody Common Targets

Secondary antibodies are commonly generated with specificity against whole immunoglobulins (IgG, IgM, IgA, IgD, IgE) and against specific antibody fragments (e.g. Fc, mu, alpha-Heavy) as outlined in the table. IgG is the most common primary antibody type. However, the other immunoglobulin isotypes and their fragments are important secondary antibody targets.


Commonly used abbreviations for secondary antibody specificity. In addition to class and species specificity, secondary antibodies can be generated against specific antibody fragments (F(ab')2, Fab) or individual antibody chains (mu, gamma, kappa) and domains. The following is a list of commonly used notations that indicate the specificity of secondary antibodies:
H+L - (heavy and light chains) whole immunoglobulin (Ig) and any molecule containing those chains or domains. This generally means that antibodies produced using this method will recognize the predominant isotypes IgG, IgM, and IgA due to their conserved regions.
Fc - (Fragment, crystallizable region) heavy chain regions forming the hinge and binding sites for Fc receptors, Protein A and Protein G
Fab - (Fragment, antigen binding) heavy and light chain regions forming the antigen binding domain
F(ab')2 - heavy and light chain regions forming the antigen-binding domains as well as the hinge region
IgM - whole immunoglobulin IgM
Fc5μ - 5 connected Fc regions of IgM including the lower portion of the mu heavy chain
M - mu heavy chain (IgM class)
γ - gamma heavy chain (IgG class)
κ - kappa light chain
λ - lambda light chain
IgA - whole immunoglobulin A
alpha-Heavy - alpha heavy chain (IgA class)
IgD - whole immunoglobulin IgD
IgE - whole immunoglobulin IgE

Whole IgM

Whole IgM is comprised of five Y-shaped units connected through their Fc domains by a J-chain. The pentamer has ten heavy chains, ten light chains, and ten antigen-binding sites. Anti-IgM secondary antibodies are generated by injecting a host with purified whole IgM. The use of secondary antibodies that recognize whole IgM frequently may result in unacceptable background and lower specificity for two reasons. First, light chains are shared by all immunoglobulin types. Second, IgG tends to be the predominant species in serum and other samples. Consequently, an antibody to whole IgM tends to cross-react with IgG light chains. Quite often a better choice is a secondary antibody that reacts specifically with the unique IgM mu (μ) heavy chain or the IgM pentameric Fc5μ fragment.


IgM Fc5μ Fragment

The Fc5μ fragment consists of the interconnected bases of the five Y-shaped units. The upper portions of the heavy chains (μ chains) and the entire light chains are absent. Thermo Scientific Pierce Anti-Fc5μ secondary antibodies are generated by injecting a host with the fragment. Because these secondary antibodies recognize the IgM fragment but neither immunoglobulin light chains nor non-IgM heavy chains, their use typically results in less nonspecific binding and background signal than a secondary antibody that recognizes whole IgM molecule.


Mu Chain

Mu (μ) chains are the heavy chains that define the IgM class of immunoglobulins. Individual μ chains are univalent with only a portion of a single antigen-binding site instead of ten antigen-binding sites, as on the whole pentamer. The light chains and "J" chain are absent. Mu-chain specific secondary antibodies are produced by injecting a host with whole IgM and then absorbing the anti-serum to remove antibodies against light chains. These secondary antibodies can detect whole IgM, Fab, F(ab')2, Fc5μ, and Fcμ fragments, as well as the μ chain itself. Because they only recognize epitopes found on the μ chain, cross-reactivity with other immunoglobulin light chains and non-IgM heavy chains is eliminated. Mu-chain specific secondary antibodies typically produce less nonspecific binding and background signal than antibodies produced against whole IgM.


Immunoglobulin Light Chains

Secondary antibodies are available to specifically recognize either the kappa (ĸ) or lambda (λ) light chains of human immunoglobulins. Light chains consist of constant (CL) and variable (VL) domains, each containing about 110 amino acids. The proximal constant domain of light chains is shared by all immunoglobulins (IgG, IgM, IgA, IgE, and IgD) within a species. The terminal variable domain is involved in antigen recognition. The two types of constant regions give the light chains their designation as either kappa or lambda. Whether kappa or lambda, all light chains are bound to heavy chains through disulfide bonds and hydrophobic interactions. Knowing the type of light chain that predominates in a sample can be critical. For example, lambda chains (and some kappa subgroups) do not bind well to Protein L. Consequently, Protein L is a poor choice for the immunoaffinity purification of immunoglobulins whose light chains are of the lambda variety.


Whole IgA

Whole IgA is made of two Y-shaped units connected through their Fc domains by a J-chain. The dimer has four heavy chains, four light chains, and four antigen-binding sites. Injecting a host with whole IgA produces anti-IgA secondary antibodies. However, using secondary antibodies that recognize whole IgA often results in unacceptable background signal and lower specificity. This is because light chains are shared by all immunoglobulin types, and IgG tends to be the predominant species in serum and other samples. Therefore, antibodies to whole IgA can cross-react with IgG light chains. To increase specificity and decrease background signal, it may be necessary to choose a secondary antibody that reacts specifically with the IgA (alpha-Heavy Chain).


IgA (alpha-Heavy Chain) fragments

Alpha chains are the heavy chains that define the IgA class of antibodies. Individual alpha chains are univalent with only a portion of a single antigen-binding site instead of four antigen-binding sites, as on the whole dimer. The light chains and "J" chain are absent. Alpha-chain specific secondary antibodies are generated by injecting a host with whole IgA and then absorbing the anti-serum to remove antibodies against light chains. These secondary antibodies can detect both IgA1 and IgA2 isotypes. Because they only recognize epitopes found on the alpha chain, IgA (alpha-Heavy Chain) fragments do not cross-react with IgG, IgM, IgE, or IgD heavy chains, T-cells, monocytes, granulocytes, or erythrocytes. Consequently, alpha-chain specific secondary antibodies typically produce less nonspecific binding and background signal than antibodies produced against whole IgA.


Whole IgD and Whole IgE

Secondary antibodies against whole IgD and whole IgE are less common, but are available. IgD and IgE are monomeric antibodies with two Ig light chains and two class-specific heavy chains, delta (δ) for IgD and epsilon (ε) for IgE. Injecting a host with whole IgD produces anti-IgD secondary antibodies, while injection with IgE produces anti-IgE secondary antibodies.


Choosing a Secondary Antibody

In particular methods, typical secondary antibodies are either too specific (e.g., recognize only one host species of primary antibody) or too general (e.g., recognize whole IgG and any fragments thereof). In most cases, these limitations can be overcome by carefully designing the experimental system and choosing the appropriate secondary probe. The following considerations are useful to help choose a secondary antibody:


  1. Determine the host species of the primary antibody (mouse anti-tubulin,rabbit anti-CD4, etc.)
  2. Select an appropriate host species for the secondary antibody (goat anti-mouse IgG, donkey anti-rabbit IgG)
  3. Consider cross-reactivity or specificity issues of the secondary:
    • Cross-adsorbed - for multiple-labeling applications or when using samples with endogenous antibodies
    • Specificity - binds to correct fragments, classes or chains of the primary antibody
  4. Detection or purification method
    • Label - appropriately conjugated to the correct enzyme, tag or fluorophore for the chosen detection method
    • Ability to bind to Protein A, Protein G or Protein L - make sure the secondary antibody chosen has sufficient affinity for the molecules used upstream or downstream (i.e., Protein A-coated microplates.)
  5. Consider requirements of the supplied secondary: Supplied state - sterile liquid or lyophilized, suspended in PBS or Tris buffer, contains carrier proteins such as gelatin or albumin or the addition of stabilizers such as sucrose or microbial inhibitors

Once these considerations have been taken into account, it may be helpful to understand how Thermo Scientific Pierce Antibodies are presented including both what the secondary antibody is and what it targets. This example may be helpful in distinguishing the structure and specificity of a particular secondary antibody as found in the Thermo Scientific Pierce catalog:


F(ab')2 Goat anti-Mouse IgG-F(ab')2 (Cross-Adsorbed) Antibody, DyLight 550 Conjugate


This name describes a goat IgG that has been purified to the F(ab')2 fragment. It targets mouse IgG and is specific to the F(ab')2 region. It is also cross-adsorbed against serum proteins of other species and is conjugated to Thermo Scientific DyLight.


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