NEW PROCEDURES FOR PREPARATION AND ISOLATION OF CONJUGATES OF PROTEINS AND A SYNTHETIC COPOLYMER OF D-AMINO ACIDS AND IMMUNOCHEMICAL CHARACTERIZATION OF SUCH CONJUGATES

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-A'-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. © 1979, American Chemical Society. All rights reserved.