Antibodies to interleukin 3 as probes for the interaction of interleukin 3 with its receptor.
A series of antibodies, directed against murine interleukin-3 (IL-3) or synthetic peptides corresponding to portions of the IL-3 sequence, has been used to detect receptor-bound IL-3 on the surface of cells. An assay was developed in which the bound primary antibody was detected using a biotinylated secondary antibody and fluorescein isothiocyanate-labeled streptavidin, followed by analysis on a fluorescence-activated cell sorter. The fluorescence signal was shown to be specific for cells known to express IL-3 receptors and was dependent on the preincubation of cells with IL-3 under conditions that did not allow internalization of receptors. Antisera raised against full-length synthetic IL-3 or to the N-terminal 29 residues were found to give equivalent signals. On the other hand, antibodies to residues 91-118 showed no signal in this assay, despite being able to bind to IL-3 in solution and neutralize IL-3 bioactivity. When peptides corresponding to residues 30-43 and 123-140 were incubated with the anti-IL-3 antiserum, the majority of the fluorescence signal was abolished, indicating that these two peptides contained the primary epitopes being recognized by the antiserum in this assay. This antiserum also bound to the 91-118 peptide, but the corresponding peptide was not able to reduce the fluorescence signal in a similar competition assay. These results suggest that the 91-118 region is not accessible to antibody when IL-3 is bound to its receptor, whereas at least portions of epitopes 1-29, 30-43, and 123-140 remain accessible to antibody.(ABSTRACT TRUNCATED AT 250 WORDS)
Duronio
V
V
Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Granleese
S R
SR
Clark-Lewis
I
I
Schrader
J W
JW
Ziltener
H J
HJ
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001667:Binding, Competitive; D015415:Biomarkers; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D007377:Interleukin-3 / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D016185:Receptors, Interleukin-3 / Q000378:metabolism*; D013696:Temperature
D000906:Antibodies; D015415:Biomarkers; D007377:Interleukin-3; D016185:Receptors, Interleukin-3