sandwich ELISA

Jackie E. Kylander jek at med.unc.edu
Thu Jan 23 12:22:00 EST 1997


In article <32e5f1f2.9272658 at news.univie.ac.at>,
a8803349 at unet.univie.ac.at (Martin Offterdinger) wrote:


>My questions are :
>
>1 Will it be helpful to use the polyclonal antibody as the
>primary(coat it to the plate) ?

Rabbits are notorious for producing nonspecific staining. In the past,
I've gotten around it by using a monoclonal to coat and a biotinylated
monoclonal to detect, then used ABC labeled with enzyme of choice,
followed by substrate. This works well if you know the exact epitope
you're fishing for and if the substance in mind has TWO such sites (one to
bind with the Ab on plate and one to bind with Ab in solution).

>
>2 What detergent should be used and in which concentration to
>solubilise the cells??-At the moment I use 0.5% digitonin in isotonic
>buffer.

Used to use .05% Tween 20, but others work, too. Have no idea of "best"
one to use.
 
>Do you think that different detergents would work better???

-- 
Jackie E. Kylander <jek at med.unc.edu>
A/OSB
MAG
TCS
Mac Nanny



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