title: Negatively charged polymers protect antinuclear antibody against inactivation by acylating agents.
abstract: For many practical applications, monoclonal antibodies must be chemically modified without any significant loss in their immunoreactivity. In some situations, however, the amino acid residue crucial for antibody activity may be highly reactive toward the modifying agent, which results in antibody inactivation. The method to prevent inactivation of a modification-sensitive antinuclear monoclonal antibody by acylating agents was developed. The method is based on the hypothesis that a highly reactive amino group exists within, or in the vicinity of, the binding site of the antibody, providing crucial interaction with negatively charged moieties of DNA. It has been shown that negatively charged polymers, such as dextran sulfate or heparin, may provide temporary protection, presumably interacting noncovalently with this amino group and thus masking it. The protecting molecule can be removed later by chromatography on a protein A column, thus regenerating modified but not inactivated antibody in the free form for use in subsequent applications. In particular, we have modified antibody 2C5 with a chelating agent, diethylenetriaminepentaacetic acid (DTPA) without the loss of activity. Modified antibody was labeled with radioactive isotope, (111)In, via chelation by antibody-attached DTPA. The labeled antibody was shown to demonstrate the same specificity of binding to nucleosomes as the nonmodified antibody, so it may be used in immunoscintigraphy or biodistribution studies. The method might be useful for the modification of other modification-sensitive antibodies with other acylating chemicals, such as crosslinking agents or biotin derivatives.
authors.affiliation: Procyon BioPharma, Inc., 1650 Trans Canada, Dorval, Quebec, H9P 1H7, Canada.
authors.forename: G P
authors.initials: GP
authors.lastname: Samokhin
authors.forename: D A
authors.initials: DA
authors.lastname: Mongayt
authors.forename: L Z
authors.initials: LZ
authors.lastname: Iakoubov
authors.forename: T S
authors.initials: TS
authors.lastname: Levchenko
authors.forename: V P
authors.initials: VP
authors.lastname: Torchilin
mesh_terms: D000215:Acylation; D000818:Animals; D000974:Antibodies, Antinuclear / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D000918:Antibody Specificity; D002614:Chelating Agents / Q000737:chemistry / Q000378:metabolism; D016264:Dextran Sulfate / Q000737:chemistry / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D007205:Indium Radioisotopes; D051379:Mice; D004369:Pentetic Acid / Q000737:chemistry / Q000378:metabolism; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D055672:Static Electricity; D013388:Succinimides / Q000737:chemistry / Q000378:metabolism
chemical_list: D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D007205:Indium Radioisotopes; D011108:Polymers; D013388:Succinimides; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D004369:Pentetic Acid; D006493:Heparin; D016264:Dextran Sulfate