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Institute of Pathology, Case Western Reserve University, Cleveland, OH 44120.
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Abstract |
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Introduction |
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Many investigators have studied Fas ligand by flow cytometric analysis of cell surface molecules and of total cellular expression after permeabilization of the membrane. mAbs have been successfully used in these analyses (1, 2, 3, 4, 5). Santa Cruz Biotechnology (Santa Cruz, CA) has produced polyclonal rabbit IgG raised against a C-terminal peptide (extracellular domain) from human Fas ligand. The company has indicated in their catalogue that this reagent specifically detects human Fas ligand by Western blot analysis and by immunohistochemistry.
In the past year several groups of investigators have reported flow
cytometric analysis to assess cell surface expression of human Fas
ligand by using the rabbit anti-Fas ligand peptide IgG from Santa
Cruz Biotechnology (6, 7, 8, 9, 10, 11). We have tested our Fas ligand expressing
transfected cell line KFL by flow cytometry with the Santa Cruz
Biotechnology polyclonal rabbit anti-Fas ligand IgG (C-20), and we
found that this Ab did not detect Fas ligand on the cell surface (Fig. 1
). Cell surface Fas ligand was verified
by flow cytometric analysis using two specific mAbs: NOK-1 and Alf-1.2
(Fig. 1). Alf-1.2 is a murine mAb produced in our laboratory by
inoculating BALB/c mice with a soluble, active form of Fas ligand
produced in Pichia pastoris (2). Cell surface expression of
Fas ligand by KFL cells was also verified by a sensitive activity assay
(Table I and Refs. 1 and 2).
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A) or by an activity assay (Table I), and
they do not possess Fas ligand mRNA as assessed by RT-PCR (2).
Nevertheless, C-20 stained unstimulated Jurkat cells (Fig. 1A). This staining was not inhibited by the presence of
soluble Fas ligand, although the soluble Fas ligand did inhibit
staining of KFL by NOK-1 (Fig. 1B) or Alf-1.2 (data not
shown). Moreover, cells from an allogeneic T cell line rested in IL-2
for 2 wk have no Fas ligand activity but were positive for staining
with C-20 (Fig. 1A). As with the Jurkat cells, this staining
was not inhibited by soluble Fas ligand (Fig. 1B). We have concluded that the Santa Cruz Biotechnology polyclonal anti-human Fas ligand IgG (C-20) is not appropriate for flow cytometric analysis of human Fas ligand expression. It should be noted that our analysis does not suggest that this immunoglobulin cannot be successfully used for the detection of Fas ligand by Western blot analysis or by immunohistochemistry. It seems reasonable to assume that the rabbit polyclonal antibodies raised against human Fas ligand peptides recognize the molecule after denaturation during SDS-PAGE for Western blot analysis or during permeabilization for immunohistochemistry but does not recognize the native configuration of the molecule that is found on the cell surface. The polyclonal rabbit IgG appears to cross-react with molecules on various human cells, but this cross-reaction does not correlate with Fas ligand expression.
Several groups of investigators have produced their own rabbit polyclonal Abs to peptides from human Fas ligand and have used these Abs for flow cytometric analysis (12, 13, 14). Although we did not analyze these other Ab preparations for utility in flow cytometric analysis, we believe it is prudent to include additional controls beyond preimmune rabbit IgG in these experiments or inhibition of staining with the peptide used for immunization to ascertain the validity of the results.
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Footnotes |
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Received for publication January 29, 1998. Accepted for publication February 24, 1998.
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