very low OD values - (Mar/11/2010 )
so, i ran two elisa plates yesterday and the results are almost identical: standards not working or showing any kind of curve, all results with OD of 0.05-0.08. Extremely low values.
Basically, I added the HRP, incubated for ~30 min (as directed - I am using a commercial ELISA), then added substrate. Absolutely no color change in the first 30 or even 45 minutes (assay directions said 15 minutes until addition of stop solution). I waited even more, and around 1 hr I was seeing some blue color. I am using TMB, by the way.
Then I added the stop solution, it turned yellow.
Ran the plates. Standards not working, thus I have to conclude that I did something wrong in my assay.
So what could it be? Should I have incubated the HRP more?
One thing I wasn't sure about: when removing a solution from the wells (e.g. detection antibody, coating buffer, etc.) do you pipette out with a multichannel pipette or do you flick the plates over a sink and then blot on absorbent paper? My protocol from the manufacturer did not mention the method for this so I pipetted. I'm not sure if this disturbed the antibodies or otherwise contributed to my lack of results.
As for my washing, I washed about 5x in between each step. Added 250 uL PBS with 0.05% tween 20 to each well, let soak for ~30 sec, flicked over sink and blotted.
Read at 450 nm.
I am a newbie to ELISA, so if you have any advice or explanations please let me know! thanks
well i ve not done much elisa s but wat i understand is you have a kit... but still u coat the antibodies??!! so it is not a coated plate with the kit.. so the problem can be anywhere from coating to detection.
As for the washing and aspiration is concerned, I generally follow either one of then, but making sure than I use the same thing in all the steps... and if aspirating I take care in not doing it too harshly...
Why not contact the kit manufacturer? they have the most experience with the product you are using.