human serum in ELISA - (Jan/11/2010 )

i will test antidrug antibody using ELISA. when i use monkey serm to do the acid dissociaton,the results were good.but when i change to human serum,it did not work. i tried many conditions.the result were almost same.
it seems something in the human serum interfer the function.
i don't know what i can do,please help.

Could you provide us with a brief protocol? It's hard to tell what the problem could be with this little information.

Gerard on Jan 11 2010, 11:32 AM said:

fine,a brief potocol
1.coat drug on a 96well plate, overnight
2.block plate
3.prepare positive control sample
rabbit serum(positive) spike into monkey/human serum, incubate 30min
4. acid dissociation 1 hr
5. neutralize with 1M Tris and add biotinylated -drug, incubate 1 hr-3hr.
6.add streptavidin-ap
7.pNPP
8.read

This potocol works well in the monkey serum.After acid dissociation,reading will be double than the non acid dissociation samples.but in the human serum
i think that there are some molecules in the human serum inhibited acid dissociation/neutralization.but how to get rid of them.

My first idea would be is it possible that human serum is positiv for your drug?
The blocking reagens is not giving aspecific binding to human serum?

Gerard on Jan 11 2010, 01:05 PM said:

It is impossible because the human serum is health people's
please give me some advices about the aspecife binding blocking reagents.

Can you tell us if your drug is a small molecule such as a digoxin (Therapeutic Drug), drug of abuse (ie amphetamine), steroid, or a large protein molecule such as TSH? I want to make sure your format and approach are correct.