Checking titres with homemade ELISAs

Goal: Check immunized rabbit serum for IgG against antigen of interest.

1. Coat Nunc maxisorp plate with antigen O/N at 4 degrees.
2. Block for 1h, probe with pre or post-immunization serum at various dilutions (1:25 to 1:10,000).
3. Detect using goat anti-rabbit IgG conjugated with AP at 1:5000 dilution for 1h.

My antigen of interest is prone to precipitating, and is the most stable in sodium citrate buffer, pH 5.5. I confess that I don’t really understand how ELISA plates work on a chemical level, but the low pH didn’t seem to affect antigen binding, as it worked beautifully.

The AP-conjugated goat anti-rabbit IgG was over a decade old, and I was worried about it not working. As a positive control, I coated 2 rows with rabbit polyclonals. 1 row was a commercially bought polyclonal against dynamin III, the other row was serum from a rabbit immunized with some sort of phosphatase. I used a ~1:500 dilution of both in PBS.