Used these a fair few times 4-5 years ago. Sounds a bit different to the protocol I used. Perhaps its a kit-dependent thing. Fairly sure the reagent diluent 1 contains bovine serum for blocking.

5% tween 20 is high but these guys are pretty good. I suspect its some oddity they needed for your particular protein of interest.

Normally volumes are pretty good. How close is it?

The kit worked super well! I followed everything as suggested and, obviously, the guys know what they are doing.

Thank you,