In article <6bpu2e$v$1 at orthanc.reference.com>,
<URL:mailto:ptaylor at medicina.ivic.ve> wrote:
> I'm trying to solve the problem of IgM instability in serum samples. We
> bring in serum samples from the field often without ice, never mind
> frozen, and I'm worried about loss of specific IgM activity when the
> samples are put into an ELISA. I understand that IgM is also lost on
> prolonged storage at 4oC. Is the problem principally aggregation? In that
> case does diluting the serum help? Is the problem proteases? Then add
> anti-proteases? J chain instability? Basically, what is ingredient X that
> I can add to serum samples to preserve IgM?
>> Thanks
>> Peter Taylor
>> Centro de Medicina Experimental
>> Instituto Venezolano de Investigaciones Cientificas
>> Venezuela
>IgM antibodies can be a problem because many of them behave as
cryoglobulins ie. they aggregate and precipitate in the cold and they also
are euglobulins ie they aggregate and precipitate at lowered ionic
strength. The problems may be more acute with antibodies of particular
V-region families which thus might also correlate with antibodies of
particular specificities. I can't see any harm in your idea of adding
anti-proteases and you might also like to add chelating reagents such as
EDTA to remove Ca++ and Mg++ which would be involved in activation of some
of the serum proteases. Apart from that I can't offer much more advice.
In my research I've always found IgMs to be a real pain to work with so
I've concentrated on IgG! I remember at the start of my PhD many years ago
being given the job of purifying an IgM monoclonal. I was using gel
filtration and it took me a long time to realise that all the IgM was
precipitating in the buffer at the top of the column!
Mike Clark,
Antibody Structure and Function Web Site is at
<URL:http://www.path.cam.ac.uk/~mrc7/mikeimages.html>
--
o/ \\ // || ,_ o M.R. Clark, PhD. Division of Immunology
<\__,\\ // __o || / /\, Cambridge University, Dept. Pathology
"> || _`\<,_ // \\ \> | Tennis Court Rd., Cambridge CB2 1QP
` || (_)/ (_) // \\ \_ Tel.+44 1223 333705 Fax.+44 1223 333875
