Monoclonal antibodies as structural probes for oligomeric human interferon-gamma.
Monoclonal antibodies (MAb) B1 and B3, specific for human interferon-gamma (IFN-gamma) failed to immunoprecipitate heat-inactivated human IFN-gamma in solution. However, both MAb retained some reactivity with denatured IFN-gamma immobilized on vinyl plates. The two MAb have been employed in a sensitive immunoradiometric assay (IRMA). In this IRMA one MAb was bound to polystyrene beads and used as immunoadsorbent. The second MAb, labeled with 125I, was used as the tracer to quantitate the amount of IFN-gamma bound to the immobilized MAb. Addition of unlabeled MAb B1 did not inhibit the binding of 125I-labeled MAb B3 (and vice versa), indicating that the two MAb react with two different and nonoverlapping epitopes. Yet, when the same MAb was used in IRMA as both immunoadsorbent and tracer, the amount of labeled MAb bound to a given concentration of natural or E. coli-derived recombinant human IFN-gamma was very similar as with two different MAb, indicating that a single IFN-gamma molecule must have two or more identical binding sites for each of the two MAb. These findings show that biologically active natural and recombinant human IFN-gamma exist in oligomeric form.
Le
J
J
Chang
T W
TW
Liu
V
V
Yip
Y K
YK
Vilcek
J
J
D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004686:Encephalomyocarditis virus / Q000187:drug effects; D000939:Epitopes / Q000032:analysis; D006358:Hot Temperature; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis / Q000276:immunology* / Q000494:pharmacology; D011489:Protein Denaturation; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007371:Interferon-gamma