problem with an ELISA

Christiane Devaux devaux.c at jean-roche.univ-mrs.fr
Mon Jan 3 11:32:33 EST 2000


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°C
4. Wash 4 times with PBS-tween 0,05%.
5. Antigen (5671,5 Da) distribution with human plasma (from 10-3 à 10-10
M) - 100µl/well. Time : 2h at 37°C.
6. Wash 4 times with PBS-tween 0,05%.
7. Polyclonal antibody (1/1000). 100µl/well. Time : 2h at 37°C.
8. Wash 4 times with PBS-tween 0,05%.
9. Antibody/enzyme conjugate (peroxydase) 1/2000 - 100µl/well. Time : 1h
at 37°C.
10. Wash 4 times with PBS-tween 0,05%.
11. Enzyme substrat OPD (+ 5µl H2O2  30 %). 100µl/well.
12. Read absorbance (450nm - non-stop reaction).


diluent citric acide / sodium phosphate :
7,3g citrique acid (C6H8O7) + 23,88g bivalent sodium  phosphate
(Na2HPO4).
Q.S.P. 1L H2O, PH 5.

OPD : o-phénylènediamine.
PBS : Posphate buffered saline.

Human plasma samples : use sodium citrate (0,129M). First centrifugation
: 15mn at 2000rpm (collect surface soluion), second centrifugation :
10mn at 3000rpm (collect
surface soluion). The human plasma is used 1 hour after sampling.

Thanks for your answer.
ERIC






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