CTL and anti-class I Abs

antibody antibody at bii.ch
Thu Dec 1 16:04:42 EST 1994


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

******************************************************************

Joerg Kirberg                     EMAIL: joerg.kirberg at bii.ch
Basel Institute for Immunology    FAX:   41-61-605 13 72
Grenzacherstr. 487                PHONE: 41-61-605 12 77 (lab)
Postfach                                 
CH-4005 Basel

Switzerland



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