Antibodies to Congo Red Dye as a Promising Tool for Detection and Destruction of Amyloid

Antibodies to azo dyes of the Congo Red (CR) type, which bind to amyloid fibrils both in vitro and in vivo, were obtained in order to develop an approach to therapy of amyloidoses based on the use of antibodies to amyloid components. Since CR and its analogues do not have readily activated groups necessary for covalent binding of these dyes to protein antigens, azo dye containing a fragment of a symmetric CR molecule and a free carboxyl group was synthesized. This compound, obtained from para-aminobenzoic acid and naphthionic acid, is 4-(1-amino-4-sulfonaphthyl-2-azo)benzoic acid (ASABA). It was shown that ASABA can be activated by a mixture of N-hydroxysuccinimide and 1,3-dicyclohexylcarbodiimide and then covalently attached to proteins. Conjugates of ASABA with king crab hemocyanin (HC–ASABA) and bovine serum albumin (BSA–ASABA) were synthesized. HC–ASABA was used as an antigen for rabbit immunization. Polyclonal antibodies from rabbit blood sera recognized ASABA in the conjugates, as well as free CR and its fluorene-based analogue, 2,7-bis(1-amino-4-sulfonaphthyl-2-azo)fluorene. These antibodies can be used for further work on the immune destruction of amyloid stained with CR type dyes.