New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates

Fu-Tong Liu, Mark Zinnecker, Toshiyuki Hamaoka, David H. Katz

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325 Scopus citations

Abstract

Conjugates of small haptens and a synthetic copolymer of D-glutamic acid and D-lysine (D-GL) have been shown to be very effective in inactivating hapten-specific B lymphocytes that bind determinants attached to D-GL. In order to extend the D-GL tolerance system to more complex protein antigens which are directly related to various human diseases, it is necessary to develop techniques for preparing stable conjugates of protein-D-GL which can then be isolated in pure form. Using hen egg ovalbumin (OVA) as a prototype protein, herein we describe conjugation and purification methods which involve (1) application of a recently developed coupling method employing m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as the coupling reagent, i.e., reaction of MBS-modified OVA with thiolated D-GL molecules generated in situ from acetyl-S-D-GL by hydroxylamine, and (2) introduction of biotin moieties into D-GL molecules to allow application of an avidin-biotin system for affinity chromatographic purification of conjugates. The reactions and isolations are carried out under mild conditions and in good yields. The conjugate prepared in this way retained a majority of antigenic determinants of OVA and was devoid of any nonconjugated protein, protein dimers, and oligomers which could pose a serious detriment to the effectiveness of tolerance induction. These methods were also applied to the preparation of D-GL conjugates of insulin. The conjugates were characterized by immunoprecipitin reactions and radioimmunoassays. The potential applications of these methods are discussed.

Original languageEnglish (US)
Pages (from-to)690-697
Number of pages8
JournalBiochemistry
Volume18
Issue number4
StatePublished - 1979
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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