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Title:Cryoprecipitation Properties and Structural Analysis of a High Affinity Anti-Fluorescein Immunoglobulin-M Antibody
Author(s):Dombrink-Kurtzman, Mary Ann
Doctoral Committee Chair(s):Voss, Edward W., Jr.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Microbiology
Health Sciences, Immunology
Abstract:A high affinity (K$\sb{\rm a} = 2.9 \times 10\sp $ M$\sp{-1}$) murine monoclonal anti-fluorescein IgM antibody (18-2-3) exhibiting low temperature insolubility in the absence of bound ligand has served as a model to study cryoprecipitation. Insolubility of 18-2-3 at low temperature (4$\sp\circ$C) had been shown to be reversible at higher temperatures 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 temperature. Procedures were developed for production and isolation of the monomeric subunit (IgM$\sb{\rm s}$) and Fab and (Fc)$\sb5$ fragments. Electrostatic interactions were implicated as being responsible for atypical low temperature insolubility of 18-2-3 since self-aggregation was sensitive to pH, ionic strength, temperature and protein concentration. Results indicated that 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.
The complete V region sequences of 18-2-3 were determined by Edman degradation and nucleotide sequence analysis. The V$\sb{\rm H}$ region of 18-2-3 was encoded by a gene (V$\sb{\rm H}$IB) of the Q52 V$\sb{\rm H}$ family that had 96% homology with that of anti-oxazolone antibody NQ7.5.3, but appeared to utilize a larger D region (D$\sb{\rm Q52}$ plus N region). The V$\sb\kappa$ region of 18-2-3 was encoded by a gene (V$\sb\kappa$IV) that had an amino acid sequence 97% homologous to that of anti-oxazolone antibody NQ11.1.18. J gene usage was J$\sb{\rm H}$4 and J$\sb\kappa$5. This was the first variable region sequence of a murine IgM that self-aggregates at low temperature.
Solid phase analyses showed that 18-2-3 was not idiotypically related to prototypic anti-fluorescein antibodies 4-4-20 and 9-40, but that 18-2-3 could bind to other IgM antibodies, both murine and human. Hemagglutination assays indicated that 18-2-3 was not a cold agglutinin. Preferential binding of fluorescein to 18-2-3 heavy chain occurred as determined by affinity labeling with fluorescein isothiocyanate isomer I.
Issue Date:1988
Description:212 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.
Other Identifier(s):(UMI)AAI8908666
Date Available in IDEALS:2014-12-16
Date Deposited:1988

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