Binding sites of interleukin-3-mimetic monoclonal autoantibodies derived from a MRL/lpr mouse.
MRL/lpr mouse-derived interleukin-3 (IL-3)-mimetic monoclonal antibodies were examined for their binding sites. One of these five antibodies (B10, F8, F9, F12, H11), F9 interacted with the IL-3 receptor, as if it were an anti-idiotypic antibody; the IL-3-mimetic activity of F9 was blocked by a neutralizing rat monoclonal anti-IL-3 antibody. IL-3 mRNA was not detected in hybridoma F9, as analyzed by the S1 protection assay, Thus, the activity neutralized by the rat antibody is of the F9 antibody itself but not the IL-3 type. Such blocking was not observed with the IL-3-mimetic activity of the other MRL/lpr-derived monoclonal antibodies. On the other hand, the binding of all these monoclonal antibodies to IL-3-dependent cells was inhibited by each other and vice versa, as analyzed by two-color flow cytometry. This indicates that the binding sites of the five monoclonal antibodies are located so close to each other that the binding of one would interfere with the binding of any one of the others (since the binding experiment was done on ice, it is unlikely that the inhibition is due to down-modulation of the receptors). Taken together the results obtained by the enzyme digestion study, we discussed that all five IL-3-mimetic monoclonal antibodies are directed to the IL-3 receptor, but only F9 binds to the portion directly responsible for the binding of IL-3 and the other antibodies (B10, F8, F12, H11) bind to different portions, respectively, which are adjacent or overlapping to the binding site of F9.
Sugawara
M
M
Nippon Roche Research Center, Kanagawa, Japan.
Miyajima
A
A
Tanaka
H
H
Tezuka
E
E
Hattori
C
C
Ohta
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001323:Autoantibodies / Q000378:metabolism*; D001327:Autoimmune Diseases / Q000276:immunology; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D007377:Interleukin-3 / Q000378:metabolism*; D051379:Mice; D008817:Mice, Mutant Strains; D009500:Neutralization Tests; D012333:RNA, Messenger / Q000378:metabolism; D016185:Receptors, Interleukin-3 / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007377:Interleukin-3; D012333:RNA, Messenger; D016185:Receptors, Interleukin-3