Hi All,
Hoping you all might have some pointers about an ELISA protocol I am trying to run. Its based on a published protocol ( " A simple, sensitive and widely applicable ELISA for S100B: Methodological features of the measurement of this glial protein" by Leite et al)
I'm using the same antibodies and protocol as the paper but my results are very poor. The standard curve is very flat and absorbances are low even when using a less dilute standard curve (10ng/ml-19pg/ml rather than 1ng/ml-1.9pg/ml).
I've tried altering coating, detection and secondary (HRP conjugate) antibody concentrations with minimal change in results.
I guess I am just wondering what is the most common cause of my issues. THe buffers I am making? (carbonate buffer to coat, PBS + BSA and tween to wash, 50mM tris to dilute samples/stds). Or is it more likely one of the antibodies and how do I test that?
Any tips you could provide me for troubleshooting would be amazing.
Thanks so much
Have you checked for change the wavelength during OD. Go for that and find out wavelength with maximum OD.
Hello Kate,
Perhaps you write to the authors. See if they could help you with troubleshooting.
I've approached the authors and they can't find anything wrong with my protocol. The antibodies are from sigma and dako. The substrate changes colour if I just add a bit of the HRP conjugated antibody so I don't think it is that.
Thanks for the answers though
Which format you are using that is competitive or non-competitive? What is the zero optical density. Pl. provide answers to it. I will try to figure out where problem persist.
In the mean while you may go through attached paper.
With best wishes
I would double check the purity/quality of your coating antigen. Are you using the exact same type of ELISA plate? How many washes are you doing?
You can do crude testing of your antigen, with a commassie gel. The same is true for your antibodies.
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