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Radiolabeling of Immunoglobulin without Loss of Antibody Activity—Use of ¹⁴C-Phenylisothiocyanate with Human Cytotoxic Antibody¹

From the Division of Immunology, Duke University Medical Center, Durham, North Carolina 27710

Abstract

Radioiodination of immunoglobulins, particularly human cytotoxic IgG, induced a marked loss of antibody activity. Phenylisothiocyanate (PITC) readily reacts with -amino groups and the -amino groups of lysines to form phenylthiocarbamyl derivatives. Since PITC is commercially available with ¹⁴C, ³H, and ³⁵S substitutions, it provided an alternative means for labeling immunoglobulin which preserved antibody activity. There were approximately 80 PITC binding sites per IgG molecule, and ¹⁴C-PITC was bound with an efficiency in excess of 80%. With as many as 40 PITC molecules bound per IgG molecule, full cytotoxic activity was retained by both anti-HLA isoantisera and human anti-melanoma autoantisera. Even with 70 to 80 molecules of PITC bound per IgG molecule, over 80% of the antibody activity remained. Radiolabeling of antibody with ³H, ¹⁴C, or ³⁵S-substituted PITC will greatly facilitate studies of antibody binding, particularly to cell surface antigens.

Footnotes

¹ Supported by United States Public Health Service Research Grants CA 13070, AI 08897, and GM 10356 from the National Institutes of Health and by Research Grant 956-A-1 from the National Multiple Sclerosis Society.