ELISA problems - Coating and OD (Jun/26/2002 )

I am using a commercial antibody against a common household protein, which gives a highly specific and strong staining on a western blot. However, when using ELISA to quantify the amount, there is a rather high variation in the triplicates, and the OD after 30 minutes of development is quite low (around 0,02-0,05).

I coat 1ug pr. well in PBS, coat overnight at 4 degrees, blocks with nonfat milk in PBS for 1 hours, uses 1:1000 1 and 2 AB dilutions, with 4 washes between in PBS.

The antibody should be usable for ELISA, and for other antibodies the OD is higher, but the variation is the triplicates is quite high...sometimes in the order of 2 to 3 times.

You could maybe try some kind of overload test. Coat your plate with the antibody then use some kind of generic stain to stain all the antibodies on the plate, take OD's and look at the variation. If the variation is large then the plate isn't coating propery, if it is small then there could be some kind of contamination or your samples might just not be evenly distributed.