It is generally understood that binding of secondary antibody enhances the signal ..Perhaps due to binding of more than one secondary antibody per primary antibody. ? Do we know exactly how many secondary antibodies bind to one primary antibody?
In theory, two secondary antibodies can bind to a single primary antibody, since antibodies made of two identical and symmetrical parts (2 x heavy and light chain complexes). However, practically there are many factors that can inhibit the binding of two secondary antibodies to a single primary antibody. Many factors are of stearic hindrances. secondary antibodies especially when conjugated with enzymes such as HRP, adds weight and size to the antibody. In this case, two antibodies can not fit in on to a single primary antibody, especially when the primary antibody binds to an antigen in a structural scaffold. This is true even for secondary antibodies conjugated with small fluorophores when it comes to antigen antibody complex in narrow spaces. Sometimes, the secondary antibody binding sites on the primary antibodies can be very close which allows only one secondary antibody binding.
So considering these issues, I would assume, binding of secondary antibody just give the signal but not enhances it. Though if we imagine each primary antibody bound by two secondary antibodies, the doubled the signal is not significant. The signal amplification is real when a secondary antibody conjugated with an enzyme which degrades a plenty of substrates to produce a huge amount of product causing the signal amplification of more than 10 folds.
I hope my theory is good enough. Thanks
I think the first alinea of Ramachandramouli Budida's answer is correct.
However when you bind multiple molecules of the same fluorophore to the secondary antibody, you will get signalamplification.
In case of enzymeconjugates (especially for IHC) secondary antibodies are labeled with polymers thus increasing the amount of enzyms bound inderectly to each primary antibody
Roland is correct. Signal amplification can be achieved by conjugating multiple fluorophores to each secondary antibody.
Thanks for valuable inputs and I agree with your suggestions. However, I just needed any experimental evidence about the number of secondary antibodies binding to the primary.
You can demonstrate this by using affinity column with an antigen attached to the matrix and then binding of specific antibody followed by the binding of the secondary antibody. You can then quantify the eluted antibody in the gel. If you get two times of your only primary antibody control, then it is only 1 secondary antibody to 1 primary antibody. if it is three times of your control, then 1 primary antibody bound by two secondary antibodies.
I hope this is helpful to you. Goodluck.
I'm not so sure about the correctness of Ramachandramouli Budida's answer, because it does not regard the polyclonal nature of most secondary antibodies. Binding of an individual epitope on the primary antibody is limited to two secondary antibodies (one per arm). But a polyclonal secondary antibody/antiserum may find additional epitopes.
I think, the main limitation is the steric hindrance (antigen structure, antibody structure, size and number of label).
Therefore I think I remember the number of about 5 or 6 secondaries per primary antibody, although I can't find any literature on this.
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