Anti-HLA Class I MAb

John Ladasky ladasky at leland.Stanford.EDU
Wed Jun 5 17:08:57 EST 1996

In article <4p4gne$mk6 at sifon.cc.mcgill.ca>,
Rolf Loertscher  <rloert at PO-Box.McGill.CA> wrote:
>Does anyone know of an anti-HLA Class I framework mAb which does not
>cross-react with mouse cells?  We've tried W6/32, which 
>binds well to mouse lymphocytes.  Thanks!

	Exactly how are you doing this analysis?  It matters.

	W6/32 recognizes an epitope that involves both the heavy chain
and beta-2 microglobulin.  A single residue has been identified in the
heavy-chain epitope: residue 121.  If 121 is a lysine or arginine, W6/32
can bind.  The other two residues found at this position are cysteine
and alanine, which are W6/32 negative.  All human class I molecules have
121 Lys; most murine class I molecules have 121 Cys or 121 Arg.  Two 
heavy chains in the mouse have 121 Arg: H-2K(d) and H-2D(b). 

	Attempts have been made to map beta-2 microglobulin residues
that participate in the W6/32 epitope by examining chimeric molecules.
The class I heterodimer is not perfectly stable.  It is possible to swap
a cell's endogenous b2m with exogenous b2m by simply adding b2m to the
culture medium.  Previous studies of this sort suggested that the W6/32
epitope required a glutamic acid at residue 44 and a glutamine at residue
89.  All mouse b2m alleles have 44 Lys and 89 Glu, so they *should* be 
negative... even with K(d) and D(b).

	Now we get to the reason that I asked how you are doing your
analysis.  If, for example, you are peforming a flow cytometry assay with
*cultured* mouse cells, beware.  In the calf-serum supplemented medium
that you are likely using, there is plenty of *bovine* b2m, which is W6/32
positive.  The bovine b2m will displace the mouse b2m, and provide the
missing parts of the W6/32 epitope with K(d) or D(b) heavy chains.  False
positives of this nature hindered early attempts to understand what W6/32
binds.  In my own experiments, I have found it helpful to incubate the
cells in serum-free medium before analysis.

	As I mentioned above, the beta-2 microglobulin epitope was tenta-
tively mapped to residues 44 and 89.  Both of these residues are nowhere
near residue 121 of the heavy chain, leading people to conclude that W6/32
recognizes "a large, discontinuous epitope."  My own studies have suggest-
ed otherwise... the picture is really much simpler than that.  The earlier
researchers had a) errors in their heavy-chain sequences and b) too few 
beta-2 microglobulin alleles to make meaningful sequence comparisons.  Stay


- Kahn-Perles B, et. al., J. Immunology 138:2190 [1987]

- Jefferies WA and MacPherson GG, Eur. J. Immunol. 17:1257 [1987]

- Maziarz RT, et. al., Immunogenetics 24:206 [1986]

- Ladasky JJ and Parham P, unpublished results (shameless plug!)

Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology, Fairchild D-105
Keywords  : immunology, music, running, Green

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