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.
IDEC Pharmaceuticals Corporation, La Jolla, CA 92037.
S V
SV
Kaveri
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R
Halpern
C Y
CY
Kang
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H
Köhler
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