ELISA problems - No attachment of antigen (Sep/25/2007 )

We are running a test using anti-albumin to coat a NUNC Maxisorp plate (11ug/ml, 100ul/well overnight @4C, rinse w/PBS-Tween), then trying to attach a range of albumin-FITC standards at a range from 100 ng/mL to 2500 ng/mL (100ul/well at room temperature for 1 hour, rinse and read with 485/20-528/20 filter wheels). So far we have had no attachment of standard. We have double-checked with the supplier that we have the correct antibody-antigen pairs and tested the albumin-FITC standard solutions to make sure they are good. We have used two different lots of antibodies for coating plates to rule out the antibody. All solutions are diluted in PBS.

Has anyone else experienced this problem or know what may be wrong?

Thanks!

-gim-

QUOTE (gim @ Sep 25 2007, 10:35 PM)

A had a few questions and points.

Have you tried another coating buffer e.g. 0.1 M Carbonate buffer pH9? Although PBS should be OK I would have thought.

I'm guessing you don't use BSA as in the blocking buffer.

Also how much Tween 20 do you use in the wash buffer? Is it 0.05%?

The range your're using seems high capture ab of 11µg/ml and standards in the ng/ml range. Most of the commercial ELISAs I've used coated the detection ab at 1-2µg/ml and use alot lower concentrations of standards pg/ml up to 1ng/ml usually. Do you think you could be exceeding the binding capacity of the plate.

Best wishes,
Ceri

-Ceri-

hi,
i guess problem is becoz ur using anti albumin antibodies for capture but not anti FITC labelled albumin antibodies. what i mean is may be the epitopes are modified upon FITC labeling.

and i agree with ceri, i hope ur not using BSA as blocking buffer by which antibody sites will be blocked .

gud luk

QUOTE (gim @ Sep 25 2007, 10:35 PM)

-donot lie for ever-