When carrying out immunofluorescence, for a protein in a mouse test subject, why is it necessary to make the secondary antibody from an anti-mouse serum?
Best,
Sehej
Cross reaction is a great concern.
In some advanced testings such as immunohistochemistry, background staining is the big issue. A separately raised antibody from another animal has to take that role of proper conjugation to the chromogen/marker/indicator.
It is logic.. How does an organism (like mouse) generate antibodies against its own antibodies!!
The secondary antibody has to match the first Ab host; otherwise, the 2Ab would not bind the 1Ab. For example, if the 1Ab is a rabbit Ab, the 2Ab has to be any source-anti-rabbit.
Cross reaction is a great concern.
In some advanced testings such as immunohistochemistry, background staining is the big issue. A separately raised antibody from another animal has to take that role of proper conjugation to the chromogen/marker/indicator.
hi, Dr. Sehej Bindra
i suggest you read these papers
1- Introduction to Secondary Antibodies
https://www.thermofisher.com/in/en/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/antibody-methods/introduction-secondary-antibodies.html
2- How to Choose the Best Secondary Antibody
https://www.immunoreagents.com/protocols/view/17
3- Cross-adsorbed secondary antibodies and cross reactivity
https://www.jacksonimmuno.com/secondary-antibody-resource/technical-tips/cross-adsorbed-and-cross-reactivity/?pdf=2120
most home-made antibodies are obtained from rabbit, an animal whose size allows relatively simple manipulations (Ag injection, Ab recovery), the variable region of Ab being responsible of the recognition specificity ; but these Ab are undetectable by a simple way ; commercial firms have developed secondary Ab which for ex recognized all the rabbit Ab (due to the constant region of Ig which are used as Ag in a second animal species for ex goat) and with cost minimization this allows the scientific community (and teachers and students) to dispose of a cheap tool
The purpose is to avoid cross-reactivity of the secondary anti-immunoglobulin antibody with endogenous immunoglobulins in the sample, that is, for techniques that use such samples.
Because the organisim’s immunsystem won’t recognize its own protein as a “nonself” and peroduce an antibody against it.
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