What is the function of the enzyme bound to the secondary antibody?
Dear Shrikant,
if the enzyme is not linked to the antibody it will be washed away from the solution and it will not be possible to detect and quantify the target molecule.
First, we conjugate primary Ab with target molecule (and wash away the excess of Ab, those who did not bind to target molecule).
Then we add secondary Ab to link to the primary Ab (and again, wash away all the surplus).
The same is with enzyme, it conjugates with secondary Ab, and the excess we wash away.
If the question is about the purpose of using two antibodies, primary and secondary, then there are two main reasons:
The enzyme catalyzes the reaction for the conversion of a substrate, which can be detected afterwards.
In ELISA for example, HRP (horseradish peroxidase) is often coupled to the secondary antibody. When adding TMB a redox reaction will take place, reducing H2O2 and oxidizing TMB. The oxidized TMB will turn blue. Afterwards the absorbance can be measured and correlates with the amount of bound secondary antibody.
Dear shrikant,
There are many techniques of knowing the presence of the virus in human body and out of them ELISA and Western Blot are very popular Technics and what are the difference you will get from the following links
What is the difference between Western Blot and ELISA? – The ...
camblab.info › Home › 1. News
Difference Between Elisa and Western Blot
www.differencebetween.com › Health › General
Comparison of Enzyme-Linked Immunosorbent Assay, Western ...
https://www.ncbi.nlm.nih.gov › NCBI › Literature › PubMed Central (PMC)
Hopefully may be helpful to you
Hi there,
The role of the conjugated enzyme is to transform the presence of the secondary antibody into a visible signal (coloration or light emission).
Dear shrikant
Function of Enzyme is help in visualizing the antigen antibody reaction. When it bound to secondary antibodies, procedure is cost effective and increases the sensitivity of procedure.
Dear shrikant
Function of Enzyme is help in visualizing the antigen antibody reaction. When it bound to secondary antibodies, procedure is cost effective and increases the sensitivity of procedure.
In both the cases enzyme converts its substrate to product. In western blot the substrate of enzyme is used is precipitable one whereas in case of ELISA it is soluble one. Therefore you visualise enzymatic reaction end product on membrane in western blot whereas in ELISA in micro-well.
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