Photochemical linking of primary aromatic amines to carrier proteins to elicit antibody response against the amine haptens

Abstract

Two chemical methods, diazocoupling and reaction with isocyanates, are commonly used to conjugate primary aromatic amines with carrier proteins in order to elicit antibody responses against the aromatic amine haptenic group. Limitations of these conjugation techniques include the requirement for specific functional groups on the carrier protein which generally limits the degree of haptenic substitution obtainable, the many possible side reactions yielding hapten-hapten and carrier-carrier conjugates which waste valuable materials and lower desired hapten-carrier conjugate yields, and, in some cases, conjugation conditions which may denature the carrier protein (e.g., alkaline coupling conditions). We report here a photolabeling approach for conjugating primary aromatic amines to carrier protiens which avoids some of the problems of other conjugation methods and which was used to elicit antibodies against the primary aromatic amine hapten. The method described here is of general application for coupling primary aromatic amines to the carrier proteins and circumvents many of the problems inherent in the isocyanate or diazocoupling methods. 3-Azid- N-ethylcarbazole (ANEC), the azido analog of 3-amino- N-ethylcarbazole, was conjugated to bovine serum albumin (BSA), human transferrin (TR), thyroglobulin (TH), poly-(lysine·tyrosine), and poly-(lysine·phenylalanine) using standard photolabeling procedures. After photolysis, the conjugated proteins or polypeptides were separated from the unbound products of ANEC photolysis on a Sephadex G-10 column. The conjugated proteins were extracted with isobutanol which demonstrated that approximately 20% of the ANEC was covalently coupled to the protein carriers and that the larger portion of the aromatic haptens was non-covalently and hydrophobically bound to the carriers. The ANEC-protein conjugates used for immunization demonstrated a total covalently and non-covalently bound ANEC epitope density of 90 per BSA, 107 per TR and 800 per TH molecule. Rabbits were immunized with the three conjugated proteins and the production of antibody specific for the 3-amino- N-ethylcarbazole hapten was demonstrated by enzyme-linked immunosorbent assay and by inhibition studies using hapten-carrier conjugates of free hapten. The results demonstrate that antibodies against aromatic amine haptens may be raised by immunizing animals with hapten-carrier protein conjugates produced by photolabeling. Since the coupling conditions are very mild and the functional group requirements are so general (requiring only the presence of CH, NH, C=O, C=S, or SH bonds) most carrier proteins should be suitable for use in this method. Moreover, the method described is rapid and generates only small amounts of waste when compared to the diazocoupling and isocyanate coupling procedures. This coupling methodology should be useful for producing antibodies against a variety of primary aromatic amine carcinogens.