How to Choose the Best Secondary Antibody

Secondary antibodies are a powerful research tool. High quality secondaries enable you to target, identify and purify proteins with greater accuracy across a range of detection techniques. Our secondaries can be supplied unconjugated or conjugated to enzymes and fluorophores to aid detection using techniques such as Western blots, Immunohistochemistry, immunofluorescence and ELISA.

 

Advantages of using secondary antibodies:

• Enhanced sensitivity

• Increased accuracy

• Greater flexibility of detection wavelength

• Signal amplification

• No need to label your primary antibody:

  1. Reduced cost

  2. No loss of valuable primary

  3. Improved results

Selecting the correct secondary antibody for your research is fundamental to reliable, reproducible results. It is vital to select a secondary antibody that will bind to the specific species, target immunoglobulin, class or subclass of the primary antibody you are using. The decision is also determined by the detection assay being used, which drives the conjugate choice (Fluorophore Vs. Enzyme). Biorbyt's Secondary Antibody selection tool will help guide you to the best choice for optimised results.

What to consider:

• Whole or fragment (F(ab')2, Fab, Ig)

• The host species of your Primary (rabbit, chicken, goat, mouse, horse etc.)

• The class and subclass of the primary antibody (IgG/IgM, IgG1, IgG2a)

• Other sources of immunoglobulin in your assay (block buffer, endogenous IgG)

• The kind of label/conjugate needed

Whole IgG or Fragment?

In general, a whole Immunoglobulin (e.g. IgG (H+L)) can be used as a secondary antibody. These will bind to both heavy and light chains of the Immunoglobulin (IgG, IgM, IgA etc) and will give the highest signal. Sometimes, it is preferable to use a fragment. Monovalent Fab fragments can effectively be used to block endogenous IgG in tissues and to 'change' the species of a primary antibody by blocking all binding sites, only allowing a secondary to bind to the single binding site of the monovalent Fab fragment. F(ab')2 fragments have been cleaved to remove the Fc domain of the IgG molecule. These are of particular use when Fc receptors are a problem on cell surfaces such as B cells. They are most commonly used in flow cytometry to ensure the secondary does not bind to the cell surface.

Host Species of the primary

The host species refers to the species of animal in which the antibody was raised. Your secondary antibody should be raised against the host species of your primary. For example, if your primary is raised in mouse, you need to select an anti-mouse secondary. It does not matter what species your secondary is raised in unless you are doing a multiple labelling experiment, where all secondaries should be raised in the same host species.

Antibody class and subclass

Choose a secondary antibody that targets your primary antibody class and subclass. Primary monoclonal antibodies are usually generated in Mouse or Rabbit and have a specific subclass. Your secondary antibody can therefore be directed specifically against the subclass of your monoclonal primary e.g. IgG1. Having a subclass specific secondary can overcome problems of two primaries being raised in the same host when you're looking at multiple labelling experiments. An anti-IgG1 will not bind to an anti-IgG2a.

Polyclonal antibodies may belong to more than one subclass. Our primary polyclonal antibodies are usually IgG (gamma chain globulin) isotype generated in Rabbit. Your secondary antibody would need to be anti-IgG and be able to recognise heavy and light chains, anti IgG H&L.

Pre-adsorption

This is important to eliminate unwanted cross-reactivity with immunoglobulins therefore increasing specificity and reducing background signal. Some secondary antibodies are cross adsorbed so they don't recognise and react to other sources of IgG in the experimental system. These antibodies have been additionally purified to remove any immunoglobulins that bind to a specific species. This allows the production of highly specific antibodies that will not cross-react to certain specified species.

Example: 
Rabbit anti-Mouse min x Rat 
All IgG molecules that bind to Rat IgG have been removed, leaving only those that specifically bind mouse, but DO NOT bind rat.

Biorbyt's Range of Conjugates:

Labels enable you to visualise your target protein. The detection method and application you are using dictate the conjugate options available to you. Biorbyt can provide both conjugated and unconjugated secondary antibodies. Our range of conjugates covers most wavelength of fluorophore and enzymes in an excellent range of target species.

Conjugates are molecules that can be added to the secondary antibody to aid detection. Your choice is dependent on detection technique, laser availability if using Flow or immunofluorescence / fluorescent IHC and other labels within the experimental system. Immunofluorescence and flow cytometry need antibodies conjugated to fluorescent reporter labels e.g. Alexa Fluor, FITC, Qdot. ELISA and Western Blot are suited to chemiluminescent, colorimetric and fluorescence systems where enzyme linked secondaries are a good choice, e.g. horseradish peroxidase (HRP), alkaline phosphatase. Our selection tool will guide you to the best choice.