High background in sandwich ELISA? - (Sep/14/2006 )

I coated the plate with polyclonal ab (raised from rabbit), blocked with either 2% BSA, or startingblock (Pierce), added my antigen (0-50ng/ml), then HRP conjugated polyclonal ab (same ab as the capture, but linked with HRP) ranging from 0.5 -5.0 ug/ml. After addition of TMB substrates, the lowest background (with antigen) had reading of 0.4, and signal to noise ratio was less than 2. Theoretically, the background reading should be 0, because the HRP conjugated ab should not bind with unconjugated ab, right?

Is there any good suggestion? Thanks.

You will tend to get less background if you use a different secondary species from your coat ab. Also, play with the amount of coat antibody per well. I have found that this can help the background as well. So can the length of secondary antibody incubation time.

You may get a high background if you haven't washed away the secondary as it's HRP conjugated. Do you wash manually or with a plate washer? You could try increasing the number of washes. You could also try blocking with 5%BSA/PBS.

One other thing I noticed is in all the ELISAs I've used (cytokines mainly) our highes standard even during optimisation is 4000pg/ml i.e. 4ng/ml. I don't think this is your problem and I guess it depends on the antigen.

I don't think your background reading will ever be 0. I normally get 0.2-0.3 in my blank wells using commercial ELISAs. Just subtract the average blank reading from your average values for the standard curves so the blank value is 0 then get the equation of the standard curve/line and remember to subtract the average blank from the average samples values before working out the concentrations using the equation.

Ceri

what's about your positive control?
If the OD value of your positive control is high enough (higher than 2.0), you could decrease your 1st Ab and 2nd Ab.
Or try to use 10%FCS/PBS to block, or try to use PBST to wash the plate after you add antigen and 2nd Ab.