I used "JUST buffer" (20mM Sodium acetate, 20mM sodium acetate 9% sucrose, 0.01% polysorbate 20, pH 5.2) for doing my ELISA to check for FcgR2A binding of my antibodies. However, I got a very high background with my secondary Antibody control (Goat anti-human IgG F'ab HRP, 1:1000 dilution). Can anyone shed light on why this happened? Also, I need a buffer which does not allow IgG or antibodies to aggregate. I need this buffer for my ELISA.
Hi Swati,
Is your "JUST Buffer" sterile and clean ?
With acetate and sucrose, it might an interesting culture medium for some microbes.
Maybe you should try to filter-sterilize it,.
I've never used this buffer before, but as a wash buffer the ionic strength looks too low. The pH is usually low too, at keast for an ELISA. Typically you would have pH 7-5-8 (e.g Tris buffer) and at least 150mM NaCl. Detergent concentration also seems fairly low (try 0.05% or 0.1%). You might also want to add BSA to the antibody diluent (0.1% or even 1%). I'm not sure if sucrose is critical to your particular assay but it is not normally used in ELISAs and I'd remove it unless essential.
While I think the wash buffer is the likely to be the problem, it is also possible that the plate has not been blocked thoroughly.
It seems like you use this buffer for incubating your primary antibody on the plate. I wonder if with such a low pH your antibody of interest is binding to the antigens at all - maybe your background seems to be so high because you don't have "real" signals or because the secondary antibody is diluted in a "normal" buffer and thus can bind?
- Badges
- Science topic