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human serum in ELISA


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6 replies to this topic

#1 elisa2010

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Posted 11 January 2010 - 08:48 AM

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.

#2 Gerard

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Posted 11 January 2010 - 11:32 AM

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

Edited by Gerard, 11 January 2010 - 11:33 AM.

Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#3 elisa2010

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Posted 11 January 2010 - 11:46 AM

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

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

#4 elisa2010

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Posted 11 January 2010 - 11:52 AM

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.

Edited by elisa2010, 11 January 2010 - 12:37 PM.


#5 Gerard

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Posted 11 January 2010 - 01:05 PM

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?
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#6 elisa2010

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Posted 12 January 2010 - 06:04 AM

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?


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

Edited by elisa2010, 12 January 2010 - 06:05 AM.


#7 sgt4boston

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Posted 12 January 2010 - 12:09 PM

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.




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