Secondary Antibodies by Target Species

Sometimes 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 consider when choosing 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

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.