Comparison of the performance of immunosorbents prepared by site-directed or random coupling of monoclonal antibodies

Carolyn L. Orthner, Frank A. Highsmith, John Tharakan, Rapti D. Madurawe, Tulin Morcol, William H. Velander

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22 Scopus citations


The majority of methods used to prepare immunosorbents immobilize antibodies through their reactive amino acid residues. The bound antibody activity of these immunosorbents is low. Hydrazide-based matrices couple antibodies through carbohydrate chains frequently located in the Fc region. This paper reports a comparative study of the performance of immunosorbents prepared by cyanogen bromide or hydrazide immobilization methods. The experiments utilized murine monoclonal antibodies to the human plasma proteins Factor IX or Protein C. The antibodies were immobilized at low densities to beaded agarose matrices which had similar properties. The hydrazide immunosorbents had binding efficiencies which were lower (anti-Factor IX) or up to 1.6-fold higher (anti-Protein C) than comparable cyanogen bromide coupled gels. However, there was no improvement in performance due to lower recoveries of bound protein from the hydrazide gels. Control experiments demonstrated that oxidation of antibody which is required for its coupling to hydrazide gels had no effect on antibody binding to antigen. Our results indicate that, as with cyanogen bromide coupling methods, site-directed immobilization through carbohydrate residues results in a restricted ability to bind to antigen. Both monoclonals were found to contain carbohydrate in their Fab′ regions through which coupling may have occurred. The frequency of carbohydrate in the Fab region and the ability to control glycosylation at these sites are factors which may impact the utility of carbohydrate-directed immobilization of antibodies.

Original languageEnglish (US)
Pages (from-to)55-70
Number of pages16
JournalJournal of Chromatography A
Issue number1
StatePublished - Sep 27 1991

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry


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