I have been working with a 96-well ELISA, in which I perform serial dilution of 8 standards in wells of a non-binding plate from Grenier bio-one (#655901) before transferring these to the assay plate for incubation. In an effort to scale-up the assay, I am now using deep-well 96-plates (LoBind from Eppendorf #30504305), but found there is greater standard protein loss as I dilute from top to bottom standard compared to serial dilution in the non-binding plate. In other words, the signal ratio between standards prepared in the non-binding plate/standards prepared in the LoBind deep-well plate starts close to 1, then consecutively decreases to 0.5-0.6 at the lowest standards.

I found that pre-coating the LoBind plate with PBS improves signal recovery relative to the non-binding plate. I add 2 mL PBS to the LoBind plate, allow it to incubate for 2-3 hours, then aspirate and dry for 1 hour before storing at 4C O/N and using to prepare the standard the day of the assay.

1) Does anyone have any idea about why the process of pre-coating, then removing PBS improves signal? pH is consistent at 7.4.

2) When I used Gibco PBS instead of Sigma PBS, the recovery was worse. Again, pH for both PBS is 7.4.

Does anyone have any insight into why this PBS coating works and has anyone experienced differences between Gibco and Sigma PBS in other contexts?