ELISA - no-coating (Nov/22/2006 )

Hi, I have a question for you.
I have to perform an ELISA sandwich assay to measure the levels of the VEGF protein in human plasma. Someone suggested me to do the measurement in duplicate for each sample and to add, for each sample, another well for the no-coating, in wich I don't use the capture antibody, and to use the value of absorption of this well as blanck for the sample. Is it strickly nacessary doing this?
With some kits it's not possible to make the "no-coating" wells, because the wells are pre-coated; so how can I measure the background value of each sample? Is it the value of the zero point of the standard curve?
Can you suggest me some references on this argument?
Thanks

Hi I have the same problem, can you help me, please?!

To get a blank value for your ELISA you normally add 100µl of buffer/fluid equivalent to your sample. So if the sample is a lavage in PBS, use PBS or for tissue culture supernatants, use culture medium. If it's a serum/plasma, I guess you could still use PBS or maybe serum from a different species (as long as the ELISA isn't supposed to cross-react). The blank sample is usually incorporated into the standard curve (buffer used to dilute the standards on its own). It will give you an idea of the level of background i.e. what OD you get when only the capture and detection ab-enzyme conjugate are present without VEGF.
Ceri

Hi Ceri,
my doubt is that the aspecific background of plasma can be different among the samples, and, omitting the no-coating, I can't value it, but I assume that It's equal for all the samples.......

If your wells are pre-coated I don't know how you can work that out. The ELISA is a commercial kit maybe the company could help you out. I wouldn't have thought this type of background would vary too much between samples. You have a normal control group to compare your other samples to anyway. Not sure if I'm helping really.

Hi, I have just written to R&D, but none has answered me.
I'm looking for references on this argument, but it' very difficult find them...

Hi, I have just written to R&D, but none has answered me.
I'm looking for references on this argument, but it' very difficult find them...