muhero at globalnet.co.uk
Wed Jan 26 17:41:42 EST 2000
Deef Suus News <deefsuus at casema.net> wrote in message
news:388f436c$0$6087 at reader3.casema.net...
> spotsnall <katzday at tamu.edu> schreef in berichtnieuws
> 86n2o6$9mm$1 at news.tamu.edu...
> > In article <388df976$0$17235 at reader3.casema.net>, deefsuus at casema.net
> > >
> > >Hi,
> > >
> > >I have immunised a rabbit and I was wonderign what kind of titers you
> > >expect. I tested the serum and I had to dilute the serum 1:100.000 at
> > >to get a reasonable ELISA-curve (even at 1:1000000 there is till a good
> > >signal). When I dilute it less the color in the ELISA is brownish
> > >bright yellow (so way too dark).
> > >Is this normal??
> > >
> > >Suzanne
> > >
> > >
> > That sounds awfully high.
> > Are you certain that you have done all the usual
> > things like coating the plate with a non-reactive protein, adding
> > to the buffers to fill nonspecific binding sites and so on?
> > That you are not using an excessive quantity of second antibody,
> > too long, using fresh reagents and so on.
> > OTOH, you seem to think you do get a reasonable curve.
> > I'm not an expert on these assays, but I would think that if you post
> > more specifics here, the antigen; immunization scheme; coating
> > elisa procedure core info etc., someone will find an answer for you.
> antigen: immunisation peptide coupled to ovalbumin (immunisation is done
> using peptide coupled to KLH)
> immunisation scheme:
> December 15th: retreived blood (preserum)
> December 15th: 1st injection in Freunds complete adjuvant
> January 12th: retrieved first blood (serum I)
> January 12th: 1st booster injection in Freunds incompelte adjuvant
> January 20th: retreived 2nd blood (srum II)
> I trested all three samples. The serum I and II are the same height.
> Preseurm is very low.
> Coating is done at pH 9.6
> Second antibody is HRP.
> Reaction only takes place for 5 min. otherwise it is too long.
You may need to dilute the HRP anti-immunoglobulin further. (possibly a lot
further). If your development reaction is going that fast it will reduce the
reproducibility of your results unless you're very careful with the timing.
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