Follow-up on Mouse Monoclonal Subtyping Assays

[Shaun D. Black]

Dear Netters,
     You may remember that back at the end of March I posted a question as 
to why my panel of monoclonal antibodies each subtyped out to 'IgM'.  We were 
surprized that only IgM's were obtained for sixteen clones, obtained from 
mice after 60 days (with one boost).  At that point, one would have assumed 
that IgG's (in a secondary immune response) would be present.  Well, two
curious items have emerged that may be of interest to some of you.
     First, certain antigens appear to _select_ for IgM as a persistent
response.  Kathleen Hopp (khopp at post.its.mcw.edu) noted that she had 
recently done a bunch of monoclonal immunizations and found that they were 
all IgM.  Ivan Shaw (ivan at mni.lan.mcgill.ca) noted that attempts to produce 
monoclonals against certain antigens sometimes resulted in almost exclusive 
production of IgM.  Fourie Joubert (bio1 at navi.up.ac.za) agreed with the 
above observations and suggested that immunization with small quantities of 
antigen over long times might help; he also noted that class response was 
mouse strain-dependent.  For example, lysozyme loop peptide could elicit an 
IgG response in SWR mice but not in C57Bl/6 [ref: Verschoor, JA et al., 
Immunol. Lett. 17, 21-28 (1988)].  Lastly, Don (johnst67%mmc.bitnet at cunyvm.
cuny.edu) reported that his purified trypanosome protein elicited only IgM, 
and that the reason might be due to the immunosuppressive properties of the 
protein.
     Second, there is more to isotyping mouse monoclonals than meets the 
eye.  Mark Bradley (mpb at cbr.dwe.csiro.au) noted that he had used a BioRad 
kit which showed all the isotypes of mAb's to be IgM, but a kit from 
Amersham (dipsticks) indicated that this was not so.  Corrine Reimer 
(creimer at unixg.ubc.ca) reported that the isotyping kit from Serotec works 
well if it is fresh.  Well, we (after a simple assay turned into a 'thing') 
found that the anti-subclass antibodies from BioRad correctly indicated IgM
(mu heavy and kappa light) via ELISA.  However, isotyping strips from Sigma
indicated that all but one of these antibodies were IgG subtypes (one
subtyped as IgM with the Sigma reagents).  After Protein-G affinity
chromatography, further ELISAs, and Westerns, it became clear that we
have obtained the correct answers (all IgM) with the BioRad reagents.  I
might add further that Shannon Hall of BioRad's technical resources division
was _exceptionally_ helpful during this process of clarification  :-) .
Sigma, on the other hand, had no explanation for the behavior of their
subtyping strips and simply offered to refund our money.
     Hope this proves useful to some of you.  My sincere thanks to all of 
you who replied to our original question!  Cheers,    Shaun
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  = Shaun D. Black, PhD     | Internet:  shaun%jason.decnet at relay.the.net = 
  = Dept. of Biochemistry   | University of Texas Health Center, at Tyler = 
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