Secondary Antibodies as Probes
Advantage of using secondary antibodies
Secondary antibodies are used for the indirect detection of a target to which a specific primary antibody is first bound. The secondary antibody must have specificity for the antibody species and isotype of the primary antibody being used and generally has a detectable tag or other label facilitating detection or purification.
While requiring more steps than using a detectable primary antibody, indirect detection of the target antigen has the advantage of increased sensitivity due to the signal amplification from multiple secondary antibodies binding to a single primary antibody. In addition, a given secondary antibody can be used with any primary antibody of the same type and host species, making it an infinitely more versatile reagent than individual labeled primary antibodies. Secondary antibodies with specificity for the primary antibodies of common species are commercially available pre-conjugated with many of the common labels, making these detection reagents commodities.
The revised Assay Development Technical Handbook is an essential resource for any laboratory using enzyme-linked immunosorbent assay (ELISA) and related plate-based assay methods. The handbook describes the essential techniques and tools for designing and optimizing ELISA Assays. Featured products include coated microplates, standards, blockers, buffers, probe-labeling reagents, secondary antibodies and detection substrates.
Contents include: Introduction to ELISA, Selecting an ELISA Plate, Thermo Scientific Pierce Microplates, Thermo Scientific Pierce Coated Microplates, Blocking and Washing, Blocking and Washing Reagents, Detection Probes, Antibody Labeling, Choosing a Substrate, Bulk and Custom Offerings, and Recommended Reading.
Secondary antibodies may be provided in three formats: whole IgG, divalent F(ab')2 fragments and monovalent Fab fragments.
Secondary antibodies are typically affinity purified from the pooled serum of immunized hosts. In this first round of purification, whole immunoglobulins binding to the immunizing antibody are recovered and mainly consist of the ~150-kDa IgG class. Further purification with Protein A or G removes all immunoglobulin classes except IgG. Whole IgG secondary antibodies produced in the manner are widely applicable, easiest to produce and least expensive for purchase.
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 or because the probed samples contain active Fc-binding proteins (e.g., Fc receptors, 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.
The F(ab')2 fragments can then be purified by passing the digestion reaction through a Protein A or Protein G column, as appropriate, which removes any undigested IgG and Fc fragments with intact Protein A/G-binding domains. The smaller Fc cleavage fragments may be removed to some extent through dialysis and gel filtration or by affinity purification.
Mouse IgG1, the most common isotype of monoclonal antibody produced, is resistant to pepsin cleavage and requires optimized digestion with ficin. In addition, it is important to note that Protein A has low affinity for mouse IgG1. Therefore, special binding conditions are recommended when Protein A is used with mouse IgG1. Alternatively, these antibodies can be purified with Protein G, which has a higher affinity for mouse IgG1.
Some species of IgG can be enzymatically digested with papain to cleave antibody between the antigen binding domain and hinge region and 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.
Producing Fab fragments with immobilized papain makes enzyme removal simple. Because papain digestion does not produce small cleavage products, the intact Fc fragments and any undigested IgG can be removed by passing the digestion reaction through a Protein A or Protein G column, as appropriate.
Ficin digestion is the recommended treatment to remove the Fc portion of mouse IgG1 antibodies.
- Tech Tip #34: Binding characteristics of Protein A, Protein G, Protein A/G and Protein L
Secondary antibodies are generated by immunizing a host animal with the antibody(s) from a different species. For example, anti-mouse antibodies are raised by injecting mouse antibodies into an animal other than a mouse. Goat, donkey and rabbit are the most commonly used host species for raising secondary antibodies, but others may be available from individual suppliers.
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-adsorption (often referred to as "Highly 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.
Highly cross-adsorbed Thermo Scientific Pierce Antibodies are purified for minimal cross-reactivity with the serum proteins of specific species and indicated by the code "min x Sp", where Sp is an abbreviation for the one or more species of serum proteins against which the secondary antibody was cross-adsorbed, as indicated below; other companies often use similar codes:
In addition to class and species specificity, secondary antibodies can be generated against specific antibody fragments (F(ab')1, 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|
|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|
|μ||mu heavy chain (IgM class)|
|γ||gamma heavy chain (IgG class)|
|κ||kappa light chain|
|λ||lambda light chain|
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:
- Determine the host species of the primary antibody (mouse anti-tubulin, rabbit anti-CD4, etc.)
- Select an appropriate host species for the secondary antibody (goat anti-mouse IgG, donkey anti-rabbit IgG)
- Consider cross-reactivity or specificity issues of the secondary:
- Highly cross-absorbed – for multiple-labeling applications or when using samples with endogenous antibodies
- Specificity – binds to correct fragments, classes or chains of the primary antibody
- 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.)
- 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
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