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Antibody titers

Deef Suus News deefsuus at casema.net
Wed Jan 26 13:56:20 EST 2000

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 can
> >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 instead
> >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  protein
> 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 procedure;
> 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.

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