Mr. Vyas,

Yes, because of all parameters depend on the interaction.

Hi Gaurav, it all depends on how sensitive your detection methods are.

Binding constants (Kds) can be in the low pico molar range, but your instrumentation has to very sensitive to measure it.

It depends on your system: If you have a high-affinity binder and a poor detection sensitivity, your system is detection limited and LOD is not dependent on the binding constant. If you have a relatively poor binding affinity in combination with a high-sensitivity detection, your system is affinity limited. Only in the latter case, the detection limit will be directly dependent on the affinity of your binder.

Thank you Dr Bojidarka B. Ivanova and Dr Steingrimur Stefansson for sharing your knowledge.

Thank you Dr. Michael G. Weller for the explanation.

As from your answer i want to clear a little more of my doubt which is suppose if a system shows a low binding constant (i.e. say 10^3 range) and a good detection limit (i.e. say 0.1 to 0.5 PPB range), then i think as per your answer it is the second case that you mentioned and the LOD is directly dependent on the affinity of the binder, right ?

then does it say anything more about the system, like there is only static interaction between host and guest due to affinity and no chemical bonding or anything like that?

I am not sure if i have represented my doubt properly or not but if you can explain something about it then it will be helpful.

Thank you.

"... the LOD is directly dependent on the affinity of the binder, right ? " Yes, you got it. However,

I did not understand your second question.

Dr. Michael G. Weller ,

what i was asking is when LOD is directly dependent on the affinity of binder, then can we say anything about the interaction of host and guest?

I mean what if the guest does not interact with the host molecule through some chemical bonding (like covalent and coordinate covalent bond) and just got trapped in the cavity of host due to high affinity. or interaction is some kind of weak electrostatic interaction (like wander wall or hydrogen bonding). then is this can be the reason for low binding constant and very good LOD?

or low binding constant and good LOD kind of situation does not say anything about this kind of bonding and interaction between host and guest?

Thank you !!.

A short comment: A covalent bond is always equivalent to an infinite affinity. A "trapping" mechanism is very rare. Usually, you have a combination of hydrophobic interactions, salt bridges, electrostatic interactions, hydrogen bonds and others. A low binding constant and a good LOD seems to be very unlikely. In my experience, many binding constants reported in the literature are wrong. Their determination is far from trivial!

Thank you Dr. Michael G. Weller ,

That is really helpful.

Thanks.

Relation between binding constant and limit of detection is that , If binding constant is higher then detection sensitivity a poor.

I was curious what should be their relative magnitude? is bidning constant always higher than LOD?

This is an interesting question in analytical biochemistry. Suppose we set aside the importance of downstream instrumentation (e.g. fluorescence detection) and focus only on the contribution of the primary antibody KD. Is there a theoretical expression for the dependence of LOD on KD?

I'm thinking the LOD must always be less than the KD, that the LOD will depend on instrument noise statistics, and that the LOD should still decrease with decreasing antibody KD because the signal-to-noise ratio will be greater in the concentration range from 0.1KD to 10KD. But these are all just personal physical intuitions. Does anyone know of a paper in which the theory of ELISA LOD is developed from first principles?