Negatively charged polymers protect antinuclear antibody against inactivation by acylating agents

For many practical applications, monoclonal antibodies must be chemically m odified without any significant loss in their immunoreactivity. In some sit uations, however, the amino acid residue crucial for antibody activity may be highly reactive toward the modifying agent, which results in antibody in activation. The method to prevent inactivation of a modification-sensitive antinuclear monoclonal antibody by acylating agents was developed. The meth od is based on the hypothesis that a highly reactive amino group exists wit hin, or in the vicinity of, the binding site of the antibody, providing cru cial 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 thi s amino group and thus masking it. The protecting molecule can be removed l ater by chromatography on a protein A column, thus regenerating modified bu t not inactivated antibody in the free form for use in subsequent applicati ons. In particular, we have modified antibody 2C5 with a chelating agent, d iethylenetriaminepentaacetic acid (DTPA) without the loss of activity. Modi fied antibody was labeled with radioactive isotope, In-111, via chelation b y 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 metho d might be useful for the modification of other modification-sensitive anti bodies with other acylating chemicals, such as crosslinking agents or bioti n derivatives. (C) 2001 Academic Press.