problem with an ELISA

Linda Thurmond lmt39822 at glaxowellcome.com
Thu Jan 6 14:08:52 EST 2000


Christiane Devaux 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°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

Eric,
I have several suggestions.

First, I think you should use 2% BSA in PBS to block your plates, and
never use milk again (good to drink, but harder to publish).

Second, you must dilute the plasma at least 8-fold, and even so you will
have some (or lots of) matrix effect.  Thus, you'll need to use a blank
plasma diluent for the standards and controls.  I generally control the
serum or plasma concentration throughout the assay, regardless of how
much I might have to dilute an unknown to get into the standard range.

Third, depending upon the affinity/avidity of the interaction between
the coating antigen and the plasma analyte, you might need to try
incubation at 4oC overnight.  Extensive washing after the plasma
incubation might wash off specific signal, too.

Fourth, you might increase the PBS from 50mM to 250mM.

Good luck,  Linda




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