A monoclonal antibody against the human IL-2 receptor binds to paraformaldehyde-fixed but not viable frog (Xenopus) splenocytes.
Others have reported that a monoclonal anti-human IL-2 receptor antibody (anti-CD25) specifically binds a membrane receptor on Xenopus laevis PHA-induced and paraformaldehyde-fixed splenic blasts. In this paper, we present evidence suggesting that this binding is an artifact of membrane damage. Specifically, significant binding of anti-CD25 could only be achieved if the lymphoblasts were acid-washed and/or paraformaldehyde-fixed prior to being incubated with the fluoresceinated antibody. For example, in a representative experiment 95% of paraformaldehyde-fixed blasts, about 19% of acid-washed but not fixed blasts, but fewer than 2% of viable (untreated) blasts were positive for the CD25 epitope. Paraformaldehyde is known to alter membrane permeability. The DNA dye propidium iodide (PI) was used to demonstrate that the acid washing procedure also causes membranes to become permeable. Flow cytometric analyses of acid-washed PHA-induced splenic blasts doubly stained with the anti-CD25 antibody and PI showed that only 1.5% of the cells that were positive for CD25 did not stain with PI. Additionally, the anti-CD25 antibody, which immunoprecipitated a molecule from human lymphoblasts of between 50 and 60 kDa, did not immunoprecipitate any surface molecules from 125I-labeled Xenopus splenic blasts. Since binding of anti-CD25 to Xenopus splenic blasts appears to occur only after membrane damage, the antibody may be recognizing a cross-reactive internal epitope that is not involved in ligand binding on the cell surface.
Haynes
L
L
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, NY.
Moynihan
J A
JA
Cohen
N
N
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D003429:Cross Reactions; D005189:False Positive Reactions; D005404:Fixatives / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism; D011108:Polymers / Q000494:pharmacology; D015375:Receptors, Interleukin-2 / Q000276:immunology*; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D005404:Fixatives; D007376:Interleukin-2; D011108:Polymers; D015375:Receptors, Interleukin-2; D005557:Formaldehyde; C003043:paraform