whole/normal serum - whole/normal serum (Oct/10/2005 )

Hi

question: is whole serum the same as normal serum? if not what is the difference?
and can i use this as block when i have a sandwich elisa where the capture and detecting antibodies are from the same host?

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hi
i guess whole serum and normal serum are the same.
Why don't you blck with standard procedures in your elisa (BSA 5% or dry not fat milk 5%) ?

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I would rather avoid to use human sera to quench a human capture elisa or mouse serum with mouse antibodies etc... You never now the composition and the binding of the sera elements so it's better to avoid problems

But as Fred33 point out BSA or non fat milk are the best and cheaper way of blocking Elisa.

pesji

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I tried BSA block but the background was still too high and someone told me to use normal serum.

the elisa i use now (with the too high background) is with 2 antibodies from the same host, if i use the capture and detection ab from different hosts, can this reduce the background and if so, why?

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Well of course serum is a very complex mix of proteins with different binding capacities and so they can have a more potent blocking activity.

For example I use chicken serum to block werstern blots detected with Rabbit polyclonal Ab's and I found that the blots were much cleaner than blocking with Milk or BSA.

So yes you can try to use serum of a different organism but just be aware that it can also interact in your assay that's all

pesji

P.S I didn't ask you did you investigate the washes ? I mean you can also increase a bit the detergent in your washes increasing the concentration of the Tween20 might help also to reduce background !

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I am quit happy, i have some better results!! my background in a checkboard titration went to around 0.2 (with a ratio of 5), does not sound very good but for me it is the best i have seen so far!
i obtained this by a few alterations, now i use roundbottom wells, increased tween-20 concentration in wash buffer from 0.05 to 0.1% and let the first washstep soak for 5 minutes.
now i am going to try to increase the tween-20 a bit more and use a 10 minute soakstep.
if i use serum instead of bsa now i am afraid it will only give more problems, the background is getting better now (it is caused by the interaction first and second ab i think because if i use only these 2 ab's (no sample) then a background of about 0.2 is obtained so perhaps i can get no lower. but on the other hand if the washing and blocking gets better perhaps i can get a better ratio, 5 is nothing after all. but at least i am going the right direction now!!

i am very happy with this forum!
so your tip in increasing the tween-20 is a very good one!! till what concentration can i increase the tween-20?

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hi
usually i use 0.05% tween. I think 1 to 2% would be the max but i've never tried.
fred

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