Cryoprecipitation properties of a high-affinity monoclonal IgM anti-fluorescyl antibody.
A high affinity (Ka = 2-3 x 10(10) M-1) murine monoclonal anti-fluorescein IgM antibody (18-2-3) exhibiting low temp. insolubility in the absence of bound ligand has served as a model to study cryoprecipitation. Insolubility of 18-2-3 at low temp. (4 degrees C) had been shown to be reversible at higher temp. and in the presence of fluorescyl ligand, indicating antigen binding site involvement. The primary objectives were to isolate and identify structural component(s) responsible for insolubility at low temp. Procedures developed for production and isolation of the monomeric subunit (IgMs) involved thiol reduction and gel filtration separation in the presence of the zwitterionic detergent, CHAPS. Fab and (Fc)5 fragments generated by papain digestion were purified sequentially by gel filtration (Sephadex G-200) and affinity chromatography (fluorescein-Sepharose). A protocol for covalent labeling of Fab fragments with the fluorescent chromophore 2,5 DNS-Cl was developed in order to measure steady-state fluorescence polarization. In kinetic and equilibrium cryoprecipitation assays, nonliganded 18-2-3 IgM and IgMs demonstrated insolubility. Bound fluorescyl ligand, increased ionic strength (0.5 M NaCl) or basic pH (greater than 8.0) abrogated cryoprecipitation. Isolated Fab or (Fc)5 fragments did not exhibit low temp. insolubility. Decreased cryoprecipitation occurred when (Fc)5 fragments were added to nonliganded 18-2-3 IgM. Fluorescence polarization of 2,5 DNS Fab 18-2-3 fragments indicated lack of Fab-Fab aggregation. Results suggested that electrostatic interactions involving 18-2-3 antibody combining sites with interactive sites in the Fc region of homologous IgM were responsible for the phenomenon of cryoprecipitation.
Dombrink-Kurtzman
M A
MA
Department of Microbiology, University of Illinois, Urbana 61801.
Voss
E W
EW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000915:Antibody Affinity; D002460:Cell Line; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003080:Cold Temperature; D003450:Cryoglobulins / Q000302:isolation & purification; D019793:Fluorescein; D005452:Fluoresceins / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification; D007141:Immunoglobulin Fc Fragments / Q000302:isolation & purification; D007075:Immunoglobulin M / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D003450:Cryoglobulins; D005452:Fluoresceins; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007075:Immunoglobulin M; D019793:Fluorescein