Hi,
I did a test with a sandwich ELISA where I incubated an antigen (human serum) in a coated microtiterplate. After the incubation I transferred the human serum to a new wells and incubated that aswell. My hope was that all the antigen in the human serum would bind in the first wells and I would see barely any signal in the second wells. This, however, didnt happen. Only 60% of the antigen was bound in the first well, and this seems to follow a trend. I used multiple concentrations and it would never bind 100% of the sample. Not even in low concentrations.
Any idea why?
I think it might be the 1 hour incubation time. But I am not sure.
How long is your incubation time? You could try to incubate for longer, possibly with slight agitation. Maybe over night?
Good luck!
As Antoni and Philipp mentioned, you may need to perform the assay with different dilutions of capture antibody versus higher dilutions of serum. Also longer incubation over night at 4C may be ideal.
You may need to give thorough wash to get rid of blocking agent (BSA or milk protein) before adding the serum.
Best
Thanks for the response!
I gave it some thought and discussed it with colleagues. One of them mentioned that leptin (the antigen) is partially bound to a carrier protein. This explains why only 60% binds, the other 40% is bound to a carrier protein that blocks the binding site of the labeled antibody. I am currently incubating for 1 hour at 37 degrees to see the effects on the bound-unbound balance.
A 100% binding is rarely achieved in immunoassays. It may need up to 36 hrs to reach a maximal yield. For practical reasons, in most immunoassays shorter incubation times are used. BTW: Shaking is important. For long incubations the plates need to be sealed to avoid evaporation.
Check some points as follows:
- Temperature: 4 or 37 Celcius
- Time: over-night if 4 and 1 hr if 37
- The material of the microplate
- Pipetting when transferring the sample