Structural properties of an anti-fluorescein monoclonal IgM cryoglobulin.
Prior studies with murine monoclonal anti-fluorescein IgM 18-2-3 indicated both a high affinity for Fl (Ka = 2.9 x 10(10)/M), a relatively lower affinity for phenyloxazolone and an active site mediated cryoprecipitability in the absence of bound ligand. Active site related electrostatic interactions appeared to correlate with the low temp insolubility of 18-2-3, since auto-aggregation was sensitive to pH, ionic strength, temp and protein concn. Results of solid phase binding assays indicated that 18-2-3 complexed with murine and human IgM molecules but not with murine anti-Fl Mab 4-4-20 (IgG2a, kappa). Hemagglutination studies showed that 18-2-3 was not a cold agglutinin. Pentameric monoclonal antibody 18-2-3 exhibited a slower association rate with fluorescyl ligand relative to the rate observed with non-cryoglobulin anti-Fl monoclonal antibodies. However, Fab fragments of 18-2-3 displayed relatively faster kinetics, normally observed with anti-Fl monoclonal antibodies. The slower association rate exhibited by pentameric 18-2-3 was attributed to competitive binding between the fluorescein ligand and 18-2-3 determinants. Derivation and characterization of 18-2-3 (Fc)5 fragments indicated co-purification of Fv fragments possessing functional antigen binding sites, providing further evidence for binding of 18-2-3 Fab fragments with isologous Fc. Since 18-2-3 bound other IgM molecules, the mechanism of cryoprecipitation appeared to be an interaction of the fluorescein antigen binding site with specific Fc epitope(s).
Dombrink-Kurtzman
M A
MA
Department of Microbiology, University of Illinois, Urbana 61801.
Lacy
M J
MJ
Voss
E W
EW
D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D002460:Cell Line; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002621:Chemistry; D003450:Cryoglobulins; D019793:Fluorescein; D005452:Fluoresceins; D006384:Hemagglutination; D007141:Immunoglobulin Fc Fragments; D007075:Immunoglobulin M; D007700:Kinetics; D051379:Mice
D000911:Antibodies, Monoclonal; D003450:Cryoglobulins; D005452:Fluoresceins; D007141:Immunoglobulin Fc Fragments; D007075:Immunoglobulin M; D019793:Fluorescein