Flow cytometry

kruskal at RASCAL.MED.HARVARD.EDU kruskal at RASCAL.MED.HARVARD.EDU
Thu Jul 20 14:12:24 EST 1995


In article <13488CA4F5F at prl.pulmonary.ubc.ca>, DANDERSON at PRL.PULMONARY.UBC.CA writes:
>I am interested in performing flow analysis on murine splenocytes for 
>T-cell antigens, B-cell antigens and enteroviruses.  We have two 
>antibodies, a mouse anti-virus monoclonal antibody and a rabbit 
>anti-virus polyclonal.  The CD antigens have worked extremely well 
>under many conditions, however, we have had problems with 
>non-specific staining with the anti-virus antibodies. 
>
>The mouse MAb is excellent for staining virus, however, in murine 
>tissue the secondary gives the background.  This occurs both with 
>the conventional direct and indirect staining with respect to a labelled 
>2' anti-mouse antibody.  The non-specificity of the rabbit antibody 
>appears to be primarily in the rabbit antibody itself as there is no 
>2' Ab background staining.
>
>If anyone has any comments or suggestions, I 
>would enjoy discussing these issues.
>
>Dan Anderson (danderson at prl.pulmonary.ubc.ca)
>The University of British Columbia
>The University of Nebraska Medical Center


Many immune cells (certainly including B cells) express Fc receptors which bind
most IgGs through their Fc region.  Thus, either your first or your second
antibodies, if intact, may bind directly to the Fc receptor on the cells.  You
should use a F(ab')2 or Fab fragment.  For many second antibodies, these are
commercially available.  Sometimes you can get away with using an intact first
antibody; otherwise, you may need to make F(ab')2 fragments yourself.  Pierce
sells a nice kit for doing this. (800-874-3723).

Good luck!
________________________________
Benjamin A. Kruskal, MD, PhD
Division of Infectious Diseases
The Children's Hospital
300 Longwood Ave.
Boston, MA  02115
617-355-5152
617-355-6575 (fax)
kruskal at a1.tch.harvard
________________________________



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