Thermally Stabilized Immunoconjugates:  Conjugation of Antibodies to Alkaline Phosphatase Stabilized with Polymeric Cross-Linkers

• Published in: Bioconjugate chemistry Vol. 9; no. 3; pp. 399 - 402
• Main Authors: Bieniarz, Christopher, Young, Douglas F, Cornwell, Michael J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.05.1998
• Subjects: Alkaline Phosphatase - metabolism; Animals; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - metabolism; Catalysis; Cattle; Cross-Linking Reagents - metabolism; Enzyme Stability - physiology; Immunoconjugates - chemistry; Immunoglobulin G - chemistry; Maleimides - chemical synthesis; Molecular Structure; Polyglutamic Acid - chemical synthesis; Polymers - chemical synthesis; Sulfhydryl Compounds - chemistry; Temperature; Thyrotropin - immunology
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc980025b

A method of conjugating poly(glutamic acid) poly(phosphorothioate)-cross-linked alkaline phosphatase to maleimide-derivatized immunoglobulin is described. Intramolecular autocatalyzed cross-linking of alkaline phosphatase at 2:1 to 4:1 polymer:enzyme ratios introduced 32−68 thiolates on the surface of the enzyme. Depending on the stoichiometry of polymer to enzyme, the cross-linked alkaline phosphatase retained 75−90% of its native catalytic activity. The cross-linked thiolate-functionalized alkaline phosphatase was conjugated to maleimide-derivatized immunoglobulin. Compared to a control prepared using non-cross-linked alkaline phosphatase, these conjugates were smaller in size and more stable to heat. The enzymatic activity of the cross-linked conjugates after incubation at 45 °C and pH 7.5 for 25 days was 35% higher than those of the highest-activity control conjugates. The conjugation process could be controlled by varying the stoichiometries of poly(glutamic acid) poly(phosphorothioate), alkaline phosphatase, and immunoglobulin.