Thymus-independent antigens: the preparation of covalent, hapten-ficoll conjugates

Abstract

A general approach is presented for covalently attaching carboxyl and primary amino groups to Ficoll and other high molecular weight polysaccharides in a readily controlled manner. The polysaccharides are omicron-carboxymethylated with chloroacetate in NaOH solutions. Amino groups are next introduced by monoamide formation with ethylenediamine using a water-soluble carbodiimide. Reactions are carried out in aqueous solutions; excess reactants and by-products are removed by dialysis. Three different ways for coupling the 2,4-dinitrophenyl hapten to the amino derivative of Ficoll are described. Preliminary evidence indicates that 2,4-dinitrophenyl-Ficolls, prepared by procedures developed in this study, are potent thymus-independent antigens which can specifically stimulate B cells both in vivo and in vitro. Hapten-Ficoll conjugates should be exceptionally useful for studies of B lymphocyte activation because of the ready availability of the starting material and the ease with which a wide variety of hapten derivatives may be prepared.