isotype controls

Alice L. Givan Alice.L.Givan at Dartmouth.EDU
Wed Mar 27 15:07:20 EST 1996

Scott Simon's suggestion about using the antibody itself (unconjugated and in
excess concentration) to pre-block cells before staining with the conjugated
antibody may have two practical problems:  first, it is expensive,  as the
pre-blocking antibody has to be used in high concentration and is,  by
definition,  different for every stain in your panel.  Secondly,  it won't work 
for an indirect staining system.  Another,  more theoretical problem may be
that the unconjugated blocking antibody would block both the specific
(antigenic) and the non-specific  (eg FC) binding of your staining antibody.  
So this "pre-blocked" control may not allow you to determine what binding of
your antibody is a result of its antigen-binding site and what is a result of
its Fc-end.  The suggested  method could under-estimate the non-specific
binding that occurs.  Any thoughts on this?
Alice Givan

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