In article <ferbas.5.00122E4A at cyto.medsch.ucla.edu>,
ferbas at cyto.medsch.ucla.edu (John Ferbas) wrote:
> I have used an anti-MHC class I antibody (W632) in an attempt to demonstrate
> MHC class I restriction in 2 CD8+ CTL clones, but have had no success.
I know
> that the antibody is saturating the targets by FACS analysis. Also, the
> antibody is added before the assay and present throughout. Is there any
> alternative way to demonstrate class I restriction? Your comments are
greatly
> appreciated.
>
Hi John,
have you checked that this mAbs have been used before for blocking studies,
or if they block some alloreactive T cells? The latter assay you could easily
prepare by primary MLR with spleen cells from two strains of mice (see below).
As most of the killing activity is via class I you should use this as a
positive readout if your assay works at all.
But I know, blocking studies are difficult:
I have been trying some inhibition in killer assays with anti-CD8. In my
(poor?) hands inhibition was never more than 50% and only evident at lower
E:T ratios (e.g. for B6 anti-BALB/c killers tested on BALB/c LPS blasts at
3:1; without blocking about 55% specific lysis). To achieve this effect I
had to add about 0.08mg/ml 53.6.7 during the assay.
Good luck
jorg
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Joerg Kirberg EMAIL: joerg.kirberg at bii.ch
Basel Institute for Immunology FAX: 41-61-605 13 72
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