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I am trying to use flow cytometry to measure a cell surface antigen (NCAM
or CD56, which belongs to the Ig superfamily) on a CHO (hamster) cell line.
The primary antibody is purified from a hybridoma (Immunotech), and it is
a rat IgG2a raised against mouse NCAM. In general, there is cross
reactivity between chicken, mouse, and rat NCAM, so I have assumed that
this will also bind CHO NCAM. My secondary antibody is goat F(ab')2
anti-rat IgG (H+L) FITC conjugate (Caltag), affinity purified by column
chromatography and adsorbed to remove cross reactivity to mouse
immunoglobulins". I'm getting 100% reactivity, as expected, but when I
titer it, the reactivity keeps going up and up without ever plateauing as I
increase antibody concentration. I have not been blocking my cells, and I
have been carrying out the incubations in PBS containing 1% BSA, which I've
now been told is not a sufficient blocking reagent.
Any recommendations? Is this a bad antibody or am I getting some sort of
non-specific binding that I can avoid if I block properly? My isotype
control doesn't react, but I'm told not to trust isotype controls.
Also, it seems that most, if not all, of the flow cytometry references I've
read in the literature do not block with non-specific immunoglobulins and
use only 1% BSA in PBS. Are they all wrong, or is Dr. Stewart being overly
cautious, or is it implied that they do this?
--This may be a side issue: What does "anti-rat IgG (H+L)" mean? Will this bind to _all_ rat immunoglobulins because it reacts to the light chain? That's the impression I get from Carleton and Sigrid Stewart's comments on page 43 of "Cell Preparation for the Identification of Leukocytes" (Ch. 3, Methods in Cell Biology, v.41, 1994), quoted here:
"A typical second antibody might be labeled "FITC conjugated goat F(ab')2 anti-mouse IgG antibody (gamma- and light-chain specific, purified by affinity chromatography)." This label contains considerable information...[next paragraph]... Since this goat polyclonal second antibody was prepared using a mouse IgG affinity column, this preparation contains antibodies that are specific for the heavy chain of mouse IgG. Because all isotypes found in serum...have light chains, they will also bind this second antibody because it is also "light-chain specific". Therefore, this polyclonal second reagent is not specific for mouse IgG at all. In order for a second antibody to be specific for IgG is must have no light-chain activity. If the antibody were only heavy-chain specific the label would read 'gamma specific'. "
Are the antibody descriptions misleading information, or is it assumed that everyone already knows this? I did not, and I thought I was buying antibody that reacted only to IgG, although I don't think it really matters in my case. On the otherhand, if it read "goat anti-rat Ig", you wouldn't know for certain if it reacts to IgG, so I suppose what is written is appropriate.
Thanks, Jim
(PS: Thanks for all the help & tips from my first post entitled "BD software"! I clarified my email signature file since it apparently was misinterpreted by some as arrogance on my part. Actually, it is the title of a free jazz tune that is very inspiring to me. Sorry about any confusion.)
...truth is marching in
- Albert Ayler, a free jazz musician
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