In article <394043D0.94118DC3 at deakin.edu.au>, Lyndal Kerr
<URL:mailto:lyndal at deakin.edu.au> wrote:
> I am having problems with some Mab's we have produced and would greatly
> appreciate any advice -
>> We immunised Balb/C mice with a Pinpoint (Promega) bacterial fusion
> protein. Prior to fusion, immune sera was tested with both the pinpoint
> fusion protein and a GST fusion of the same protein. End point titres
> were excellent using both antigens, suggesting a good immune response to
> our protein of interest. Performed fusion and used the pinpoint fusion
> protein to screen supernatants. Positives were selected then
> re-screened with the GST fusion protein to identify clones secreting
> antibodies specific for our antigen and not the pinpoint tag. Two lines
> were pursued (2 rounds of single cell cloning - all OK - isotyped as IgM
> Kappa) and supernants collected for use in westerns and
> immunohistochemistry. When titering the Mab by ELISA we used the
> purified pinpoint fusion protein, GST fusion protein and the cleaved
> (GST) protein, including GST alone as a negative control. Both
> supernatants from each line showed strong immunoreactivity with all
> antigens including the GST -ve controls (all other controls - OK). This
> is bizarre since the GST fusion protein was not used to immunise mice.
> We have also performed Western and Dot Blotting with the same
> supernatants using the antigens listed above and obtained completely
> different results (Westerns - weakly detecting GST but none of the other
> antigens; Dot Blots - weakly detecting all GST contructs but strong
> reactivity with the pinpoint fusion protein). Immunohistochemistry
> using the Mab supernatants has localised our protein to a specific
> region in the brain however no staining is seen in any region of the
> brain using anti-GST!
>> I know this is rather long-winded but I would much appreciate any idea's
> - we are banging our heads against the wall!!!
>> Thanks in advance
>> Lyndal Kerr
>lyndal at deakin.edu.au>
This is not an unusual occurrence in making hybridomas. The mistake is in
thinking that you only get back monoclonal antibodies specific for the
immunising antigen. Immunisation helps to increase the probability of
getting the antibody you want but isn't an absolute condition.
I learnt a similar lesson in the early days of hybridomas when I was a PhD
student in Cesar Milsteins laboratory. I had immunised rats with mouse
immunoglobulin with the intention of making some useful reagents for
screening mouse monoclonal antibodies. I carried out the fusion and then
screened the hybridoma supernatants on SRBC coupled with mouse Ig. This was
a convenient screening system as I could look for agglutination and lysis
just as used by Kohler and Milstein for Sp1 in their 1975 Nature paper.
What I ended up with was some of the first rat monoclonal antibodies
specific for SRBC (not mouse Ig!) even though SRBC was not an immunogen.
>From phage work we that numerous antibody specificities can be obtained
from naive libraries without any need to immunise. Immunisation increases
the probability of success but is no guarantee.
In your case I suggest you need to screen more hybridomas and to include
the negative controls at an earlier stage. The fact that your antibodies
are IgM may be indicative that they are part of a primary repertoire not
selected on your immunising antigen, i.e. no class-switching to IgG has
taken place. You conduct your assays using IgG specific reagents to avoid
detecting IgM although this might restrict your selection too much if no
IgG antibodies have been made.
P.S. [ Don't forget August 2000 is the 25th Anniversary of the Kohler and
Milstein Nature paper. There is a special issue of Immunology Today in
Mike Clark, <URL:http://www.path.cam.ac.uk/~mrc7/>
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