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
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