I recently conducted a VEGF-165 ELISA using a standard curve and encountered an unexpected observation. I'm reaching out to see if anyone can offer insights or corrections to my procedure.

Here’s a brief overview of my protocol:

Interestingly, I also obtained an optical density (OD) reading for the 0 pg/ml standard. For this well, I added only BSA and followed through with the usual steps, including washing and adding TMB reagent. Despite expecting no color development, I observed some degree of color change.

Am I supposed to see any color development in the 0 pg/ml wells?