I am currently working with a set of isotype-matched murine monoclonal
antibodies (IgG1,2a, 2b, 3, M). The V regions have been sequenced to
confirm that there have been no changes in the variable regions. However, I
would like to have a dimeric IgA monoclonal (to get it secreted at mucosal
surfaces) that can be added to this set. I have received a dimeric IgA
monoclonal with the same specificity as the set (LPS -O- side chains) but
the V region DNA and protein sequences of the heavy and light chains are
different ( both VH and VL use different J region genes) than the original
set.
Therefore, it would not really be fair to use this dimeric IgA monclonal in
the set to examine the role of isotypes in immunity to a bacterial
pathogen.
I have thought of a possible solution. but would like feedback from "those
who know".
1. Try to force a class switch of the IgM monoclonal to IgA using TGF
beta.
1a. To get a dimeric IgA would require that the IgM hybridoma secretes
pentameric molecule and thus produces J chain. Are most IgM monoclonals
monomeric or pentameric ( I am in the process of running some culture sups
on a nondenaturing gel to determine this)? If it turns out to be monomeric
can you transfect a functional J chain gene-containing expression vector
and convert them to pentameric molecules. Does anybody know if such a
clone exists or is available?
1b. Does TGF beta cause class switching to IgA on its own or in
combination with another cytokine ex. IL4, IL5, IL6?
Alternatively is there an expression vector that contains the mouse IgA
heavy chain that one could clone a V region into? This might get
complicated since again we would need a J chain clone as well as a kappa
clone
Thanks for any suggestions. Email or post-any help is welcome.
Mike Preston
