Self-binding antibodies (autobodies) form specific complexes in solution.
In this report we have shown that members of the murine self-binding antibody family, S107, form soluble complexes and precipitate under conditions in which non-self-binding antibodies remain in solution. Two approaches were used to demonstrate the self-association of autobodies: size-exclusion column chromatography and polyethylene glycol (PEG)-mediated precipitation assay. The anti-phosphorylcholine antibody T15 and two somatic variants, U4, which binds DNA, and U10, which has no identified specificity, produced larger precipitates in 10% PEG than other non-self-binding antibodies. The selectivity of PEG-mediated precipitation of self-binding antibodies is demonstrated by reduction of precipitation with specific haptens known to inhibit self-binding in solid-phase assays. Phosphorylcholine and nucleotides reduced precipitation of T15 and U4, respectively, but not U10. To rule out Fc-Fc mediated self-association in solution, we have also demonstrated self-complexing of F(ab')2 fragments of T15 using PEG. The self-binding locus was further dissected using peptides derived from V regions. A 24-residue peptide derived from the second hypervariable region of the VH of S107 specifically enhanced precipitation of T15, U4, and U10, but not other antibodies. These results provide evidence of a dormant potential of self-binding antibodies to precipitate under conditions that reduce the solubility of proteins. The implication of this potential is discussed with respect to pathological complex formation.
Kaveri
S V
SV
IDEC Pharmaceuticals Corporation, La Jolla, CA 92037.
Halpern
R
R
Kang
C Y
CY
Köhler
H
H
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000276:immunology; D007130:Immunoglobulin Idiotypes / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000378:metabolism; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D051379:Mice; D011092:Polyethylene Glycols; D011485:Protein Binding; D012995:Solubility; D013329:Structure-Activity Relationship
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; D011092:Polyethylene Glycols