problem with an ELISA

stam0001 at my-deja.com stam0001 at my-deja.com
Mon Jan 3 12:55:00 EST 2000


In article <3870CF1F.3E35B897 at jean-roche.univ-mrs.fr>,
  Christiane Devaux <devaux.c at jean-roche.univ-mrs.fr> wrote:
> Hello I work in france on a national laboratory and I have some
problems
> with a kind of ELISA (sandwich). Well, I have to use human plasma in
my
> Elisa and this plasma brings a lot of non specific signal (clutter). I
> take the plasma from human blood (of the day) after 2 centrifugations
> (first at 2000 rpm during 15 mn and the second at 3000 during 10 mn).
So
> when I want to test a molecule (about 5671,5 Da) with Elisa, there is
a
> lot of clutter about 30% during the reading (OD : 450 nm).
> What did I have to change to clean my results ? I read a paper about
> dilution of plasma but I don't think it's a good idear. Is there
another
> solution ???
> So I have some difficulties with that test ELISA because I can't use
> serum : I need to use plasma to measure the degradation of the
molecule
> (5671,5 Da) by the proteases of blood. So I can't change plasma by
serum
> but I wonder if I change some parameters in that test ELISA (for
exemple
> the watch) or in the preparation of the plasma (for exemple the time
or
> the speed of centrifugation) I'll change my background.
>
> ELISA
>
> 1. Coat with monoclonal antibody- 1µg/ml- 100µL/well (Nunc, maxi sorp
TM
> surface). Solution citric acide / sodium phosphate . Time : 2h at 37°
C +
> 12h at 4°C
> 2. Wash 4 times with PBS-tween 0,05%.
> 3. Postcoat (milk 5% - 300µL/well). Time : 1h at 37°
Try using 5% bovine serum albumin for blocking rather than milk powder
Add albumin to wash solutions (1%)
>


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