title: A robust method for the preparation and purification of antibody/streptavidin conjugates.
abstract: The many uses of antibody-protein conjugates, especially antibody-streptavidin conjugates, give rise to the need for a reliable conjugation method offering reasonable yields and reproducible quality. We describe a method for preparing antibody-streptavidin conjugates that has consistently produced conjugates of quality and in sufficient quantity to be used in the clinical development and evaluation of the Pretarget delivery system. In this method antibody disulfides are reduced to generate reactive thiols, and maleimides are linked to streptavidin with the heterobifunctional cross-linking agent, SMCC. The two activated proteins are then mixed briefly before the conjugation is terminated with an oxidizing agent that reforms disulfides from unreacted thiols. The preponderance of the conjugate produced is 1:1 and 1:2 Ab:SA conjugate. This fraction is isolated from unconjugated proteins and high molecular weight byproduct by iminobiotin affinity and ion-exchange chromatography. The resulting conjugate is at least 90% 1:1 + 1:2 Ab:SA conjugate, contains no SA or Ab, and is produced reproducibly in 37% yield.
authors.affiliation: NeoRx Corporation, 410 W. Harrison Street, Seattle, Washington 98119, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: Hylarides
authors.forename: R W
authors.initials: RW
authors.lastname: Mallett
authors.forename: D L
authors.initials: DL
authors.lastname: Meyer
mesh_terms: D000818:Animals; D000912:Antibodies, Neoplasm / Q000737:chemistry* / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology; D003432:Cross-Linking Reagents / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D008722:Methods; D051379:Mice; D008819:Mice, Nude; D009374:Neoplasms, Experimental / Q000276:immunology; D019809:Streptavidin / Q000737:chemistry*; D014183:Transplantation, Heterologous
chemical_list: D000912:Antibodies, Neoplasm; D000936:Antigen-Antibody Complex; D000951:Antigens, Neoplasm; D003432:Cross-Linking Reagents; D018796:Immunoconjugates; D007074:Immunoglobulin G; D008301:Maleimides; C025285:N-(4-carboxycyclohexylmethyl)maleimide N-hydroxysuccinimide ester; D019809:Streptavidin