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Answers to frequently asked questions on secondary antibodies

What is the difference between a primary and a secondary antibody? What does the secondary antibody bind to?

 

A primary antibody links to the antigen directly, whereas secondary antibody links specifically to the primary antibody.

 

What does a secondary antibody do?

 

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 both for the antibody species as well as the isotype of the primary antibody being used. Also, a secondary antibody generally has a detectable tag or other label facilitating detection or purification:

 

-       Enzyme conjugates like alkaline phosphatase (AP)

-       Horseradish peroxidase (HRP),

-       Fluorescent conjugates like Alexa Fluor, DyLight, Fluorescein (FITC),

-       Biotin.

 

What are the advantages of indirect detection by secondary antibodies ?

 

You can of course label your primary antibody for revelation but indirect detection offer the following advantages :

 

-       increased sensitivity due to the signal amplification from multiple secondary antibodies binding to a single primary antibody

-       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 individually labeled primary antibodies.

 

Do you have some examples of secondary antibody applications ?

 

-      When working with mice on preclinical experiments for instance, it’s useful to measure mouse B immune response to a particular antigen or within a particular physiological conditions. You can then make the choice of rat secondary antibodies to perfectly target your primaries.

 

-     In order to purify mice mAbs, specific rat antibodies can be inserted on resin column to conduct immuno-affinity Down-Stream Processing capturing 99% of the desired antibody within linking undesired bovine components.

 

-    When conducting an ELISA, a rat labelled detector mAb with high affinity for a mouse capture mAb will allow to generate a measurable signal.

 

-       In order to define which isotype is secreted in animal in reaction to any particular antigen or particular diseases you can use an isotype kit based on Blot technology.

 

And if I don’t work with mice in my lab ?

 

This is why SYnAbs has developed all isotypes and anti-isotypes class and sub-class antibodies targeting the entire repertoire of mouse, rat,  human, guinea pig, rabbit, pig, horse, cow, dog and rabbit to cover a maximum of possibilities.

 

Are secondary antibodies polyclonal?

 

Not necessarily. A secondary antibody can be polyclonal or monoclonal, just like a primary antibody. Polyclonal secondary are more numerous since they allow greater signal increases during revelation and are cheaper to produce, but monoclonal offer different advantages. Guinea pig polyclonal antibodies offer a huge signal amplification for instance.

 

How do you choose a secondary antibody?

 

First step is to ask yourself is the species used to raise your primary antibody. For instance, if you have a rat monoclonal antibody targeting a human CD, you need to use a mouse anti-rat secondary antibody. But in addition to specifically target the host species you also need to target the class of the primary antibody. For example, if the mentioned rat antibody is a IgG2c, you’re going to search for a mouse anti-rat IgG2c.

 

What are the isotypes of rat antibodies ?

 

            Same as human and mouse, rat has five antibody isotypes (IgA, IgD, IgE, IgG, and IgM).

 

What is isotype control ?

 

           Isotype controls are primary antibodies that lack specificity to the target, but match the class and type of the primary antibody used in the application.

 

      Isotype controls are used as negative controls to help differentiate non-specific background signal from specific antibody signal

 

Causes of background staining can be multiple:

Ø  binding to Fc receptors on target cells,

Ø  nonspecific antibody interactions with cellular proteins, carbohydrates, and lipids,

Ø  cellular autofluorescence.

 

           Typically, an isotype control is matched to the host species and isotype of your specific primary antibody.

 

         Sometimes, it is an antibody that has been raised against an antigen that is not normally expressed in the target tissue, e.g. Dinitrophenol (DNP)When using directly labeled primary antibodies, it is also necessary to make sure that the isotype control is conjugated to the same fluorochrome or label as the test antibody.

 

 

"Bio-X Diagnostics is involved in the development and commercialization of kits for the diagnosis of infectious diseases of livestock. Our company uses for the manufacture of its serological tests secondary monoclonal antibodies produced by SYnabs. These reagents give us full satisfaction and we consider that the great diversity of the antibodies available is a strong point of the offer of SYnabs"

 

Philippe Coppe, CEO of Bio-X Diagnostics