lmmunochemistry, 1977, Vol. 14, pp. 449457. Pergamon Press. Printed in Great Britain IMMUNOCHEMISTRY OF SERUM ALBUMIN--IV IMMUNOCHEMICAL CROSS-REACTION OF FRAGMENTS FROM THE FIRST THIRD AND THE LAST THIRD OF BOVINE SERUM ALBUMIN* AHMED F. S. A. HABEEB and M. ZOUHAIR ATASSI Division of Clinical Immunology, University of Alabama Medical School, Birmingham, AL 35233; and Department of Immunology, Mayo Medical School, Rochester, MN 55901, and Department of Biochemistry, University of Minnesota, Minneapolis, MN 55455, U.S.A. (First received 9 November 1976; in revised form 3 January 1977) Abstract--Antisera were raised in rabbits against fragment 11-193 (less Arg-144) or fragment 37%571 of bovine serum albumin (BSA). Several rabbits and bleedings at different time intervals from each rabbit were studied. Fragment 377-571, corresponding to the last third of BSA, was invariably more immunogenic than fragment 11-193. Antisera to fragment 377-571 showed, in quantitative precipitin analysis, appreciable cross-reactions with BSA and with fragment 11-193, which increased with time after immunization. Similar results were obtained with antisera to fragment 11-193. The reactivity of antisera to fragment 377-571 with BSA was removed almost completely by absorption with fragment 11-193. Conversely, absorption of the antisera with BSA removed their reaction with fragment 11-193 almost entirely. Joint absorption by BSA and fragment 11-193, each at equivalence, gave a reaction similar to that obtained by either fragment 11-193 alone or BSA alone in the region of antigen excess. After absorption of antisera to fragment 377-571 with BSA, addition of fragment 11-193 at equivalence (relative to the unabsorbed antiserum) gave no immune precipitate but inhibited 100~ the precipitin reaction of the absorbed antisera with fragment 377-571. However, antibodies in these antisera were removed quantitatively on an immunoabsorbent of fragment 377-571. Similar findings were obtained in absorption studies on antisera to fragment 11-193. A BSA-immunoabsorbent removed quantitatively all the antibodies in whole antisera to fragment 377-571. Similarly, an immunoabsorbent of fragment 11-193 removed all these antibodies. Antibodies recovered from these immunoabsorbents did not react best in precipitin reaction with BSA or fragment 11-193, but with fragment 377-571. In this respect, they behaved exactly like the original whole antiserum and explained the observed differences in the precipitating ability of the antisera with fragments 11-193, 377-571 and BSA. These studies confirmed that BSA has repeating identical antigenic sites. Furthermore, fragments 377-571, 11-193 or BSA were essentially the same immunochemically but antisera directed against any of these as an immunogen differed only in their abilities to form insoluble immune complexes, and not in their abilities to bind with the other two cross-reacting antigens. INTRODUCTION In recent studies from our laboratories we have reported (Atassi et al., 1976a) the isolation, characteri- zation and immunochemistry of a fragment from the N-terminal third (sequence 11-193) of bovine serum albumin (BSA). Also, the preparation of another frag- ment from the C-terminal third (sequence 377-571) of BSA was reported (Habeeb & Atassi, 1976), together with similar detailed immunochemical studies. Neither fragment gave an immune precipitate with antisera to BSA but each by itself inhibited the reaction of BSA with its antisera almost completely (89-93~o). Also, each fragment, on an immunoabsor- bent, removed almost all (89-95~o) of BSA antibodies from the antisera. On the basis of these findings we * This is the 4th article in the series "Immunochemistry of Serum Albumin". The preceding article in the series is Habeeb & Atassi, 1976. The work was supported in part by grants AI 11974, AI 13181, and AM 18920 from the National Institute of Allergy and Infectious Diseases, and the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service. t Abbreviations used: BSA, bovine serum albumin. advanced the concept that BSA carried identical repeating antigenic determinants (Atassi et al., 1976a; Habeeb & Atassi, 1976). In the present work, this concept was subjected to further investigation by pre- paring antisera to each of the aforementioned frag- ments (11-193 and 377-571) and studying the cross- reactions of the two fragments as well as of BSA with these antisera. MATERIALS AND METHODS Materials Bovine serum albumin (4 x crystallized) was obtained from ICN Pharmaceuticals Inc. Trypsin-TPCK was from Worthington Biochemicals Corp. Fragment 11-193 was prepared by tryptic hydrolysis of citraconyl-BSA, followed by removal of the citraconyl masking-group and gel fil- tration on Sephadex G100 as recently described in detail (Atassi et al., 1976a). Fragment 377-571 was isolated from a partial tryptic hydrolysate of BSA by gel filtration on Sephadex G-100, followed by chromatography on DEAE- cellulose (Habeeb & Atassi, 1976). Each of the fragments was shown to be homogeneous by disc-electrophoresis. Im- munoabsorbents carrying fragment 11-193 or fragment 377-57l were prepared as previously described (Atassi et 449