Shigeki Sakata

Immunochemistry of serum albumin. X. five major antigenic sites of human serum albumin are extrapolated from bovine albumin and confirmed by synthetic peptides

Abstract In a recent report from this laboratory, we have predicted and confirmed by synthesis the locations of five major antigenic sites of bovine serum albumin. In view of the high structural similarity between bovine and human serum albumins, analogous regions of human serum albumin are predicted here to comprise antigenic sites in this protein. Inimunochemlcal studies with antlsera to human albumin and the synthetic antigenic sites of bovine albumin verified this prediction and also identified the major structural locations responsible for the immunochemical cross-reaction between these two albumins.

Immune recognition of serum albumin—XIII. Autoreactivity with rabbit serum albumin of rabbit antibodies against bovine or human serum albumins and autoimmune recognition of rabbit serum albumin

Abstract Recently, this laboratory reported an autoreactivity of myoglobin (Mb)‡ antisera with the Mb of the species in which the antisera are raised. Also, animals injected with autologous Mb mounted an autoimmune antibody and T-lymphocyte proliferative response against this protein. This posed the possibility that autoimmune recognition might be a general phenomenon not confined only to sequestered proteins such as Mb. Using RSA, we have demonstrated unambiguously that RSA cross-reacted with rabbit antisera to bovine (BSA) or to human (HSA) albumin. In exchange experiments, 125I-labelled RSA was bound by the IgG in the immune complexes isolated from rabbit antisera to BSA or HSA. Also, 125I-antibodies were bound by RSA-adsorbents. Both binding activities were inhibited specifically by RSA. RSA was isolated from three rabbits and each rabbit was immunized with its own RSA. In each rabbit autoantibodies were found by exchange between immune complexes and the rabbits own [125I]-RSA. Also, 125I-antibodies from each rabbit were bound by adsorbents of the rabbits own RSA. Inhibition studies, data on preimmune sera and on antisera against other proteins confirmed the specificity of the binding. The findings confirm the universality of autoimmune recognition and lend support to our previous suggestion that antigenic sites are ‘structurally-inherent’ in the protein.

GENETIC CONTROL OF THE IMMUNE RESPONSE TO HEN'S EGG‐WHITE LYSOZYME IN MICE: I. ANTIBODY AND T‐LYMPHOCYTE PROLIFERATIVE RESPONSES TO THE NATIVE PROTEIN*

We have initiated studies to determine whether the antibody and T‐lymphocyte proliferative responses to lysozyme and its antigenic sites is genetically controlled in mice. Mice of the H‐2f, H‐2k and H‐2p were high responders, while haplotypes H‐2b, H‐2d, H‐2r and H‐2s were low responders. Studies with recombinants indicated that the immune response is controlled by two H‐2I region loci, one being in the I‐A subregion and the other may be in the I‐C subregions.

Immunochemistry of serum albumin. VI. A dynamic approach to the immunochemical cross reactions of proteins using serum albumins from various species as models.

Antisera against bovine serum albumin were raised in two rabbits. Serial bleedings were obtained at different times after the first immunization, and antisera from these serial bleedings were not mixed but were kept and studied separately. The immunochemical cross-reactions of these antisera with serum albumins from bovine, goat, sheep, porcine, horse, human and chicken were determined by immunoadsorbent studies. These were done by titration so that the values of maximum (plateau) binding by each albumin of radioiodinated antibodies were determined. In each rabbit, the immunochemical cross-reactivity was not static but increased progressievly with time after the first immunization. In the interval 7 days to 398 days the increases in cross-reaction were extremely large. pH dissociation studies revealed that, together with the increase in cross-reactivity of a given albumin with time after immunization, there was a restriction in the antibody heterogeneity towards populations possessing higher affinity. These results provide a rational explanation for the different values of cross-reactivities for a given albumin from different laboratories. The findings are analyzed in relation to the antigenic structure of albumin and their significance in evolutionary studies discussed.

Immune recognition of serum albumin. 11. Mouse antibodies against bovine serum albumin recognize the same antigenic sites that are recognized by rabbit antibodies

In recent studies, we have localized and confirmed by synthetic peptides five major antigenic sites in each of bovine and human serum albumins which are recognized by rabbit antibodies against the respective protein. In order to determine whether the antigenic sites of albumin are peculiar to the protein or are dependent on the species of the immunized host, we have studied here the specificity of the antibody response in outbred mice against bovine serum albumin. By immunoadsorbent titration studies, the synthetic antigenic sites, previously localized with rabbit antisera against bovine serum albumin, were shown to bind considerable amounts of specific mouse 125I-labelled antibodies against bovine serum albumin. The amounts of mouse anti-bovine serum albumin antibodies bound by the adsorbents of the synthetic sites were comparable to the amounts of rabbit anti-bovine serum albumin antibodies that could be bound. It was concluded that recognition of the antigenic sites of serum albumin is independent of the immunized species and is inherent in their structural and conformational uniqueness.

Immune recognition of serum albumin-XIV. Cross-reactivity by T-lymphocyte proliferation of subdomains 3, 6 and 9 of bovine serum albumin

Recently, we have shown that, with rabbit antibodies against BSA, the BSA fragments 115-184, 307-285 and 505-582 (essentially corresponding to subdomains 3, 6 and 9 of BSA) exhibit a cross-reaction which increases remarkably with the time antisera are obtained after the initial immunization. In the present report, we have examined whether this cross-reaction at the B-cell level takes place also at the T-cell level. Optimum conditions for T-cell proliferation to BSA and the three fragments were determined in terms of priming dose, challenging dose, time lymph nodes are obtained after immunization and finally duration of culture. Several strains of mice representing independent haplotypes and recombinant strains were examined for their responsiveness to BSA by T-lymphocyte proliferation, performed in the respective preimmune sera of the same mice. This afforded the identification of high- and low-responder strains to BSA. It was determined that the immune response to BSA is controlled by genes in the I-A subregion of the H-2 gene complex with some slight non-H-2 influences. In order to avoid the possibility of genetic exclusion of response to a given antigenic site and to improve the change of total site recognition, two high-responder strains (B10.M and B10.G) were crossed. Antibodies raised in (B10.M X B10.G)F1 mice recognized subdomains 3, 6 and 9 and these were found to be cross-reactive. Specific 125I-labelled antibodies isolated on a given subdomain-adsorbent were bound very well by adsorbents of the other two subdomains. T-cells from the F1 mice that had been primed with subdomain 3 responded to subdomain 3 and were also high responders to subdomain 6 and intermediate responders to subdomain 9. After priming with subdomain 6, the T-cells responded equally as well to subdomains 3 or 6 and slightly to subdomain 9. Finally, priming with subdomain 9, gave T-cells that responded to subdomain 9 and also gave high responses to subdomains 3 or 6. It was concluded that the cross-reactions originally observed at the B-cell level also take place at the T-cell level.

Immune recognition of serum albumin—XII. Evidence for time-dependent immunochemical cross-reactivity of subdomains 3, 6 and 9 of bovine serum albumin by quantitative immunoadsorbent titration studies

Abstract Antisera were raised in rabbits against bovine serum albumin (BSA) or against fragment 377–571 (domain 3) of BSA. Serial bleedings were obtained at different times after immunization. Fragments 115–184, 307–385 and 504–581 were isolated in pure form from BSA. These corresponded essentially to subdomains 3, 6 and 9 respectively of BSA. The capacity of each fragment to bind 125 I-labelled antibodies of rabbit anti-BSA or anti-domain 3 antisera was determined by a quantitative immunoadsorbent titration technique. With anti-BSA antisera from each rabbit, the maximum (plateau) amount of antibodies bound by each fragment was found to increase with time antisera are obtained after the first immunization. With antisera against domain 3, considerable amounts of antibodies were bound by subdomains 3, 6 and 9 and the fraction of cross-reacting antibodies also increased with time after the initial immunization. Antibodies against each subdomain fragment were isolated on fragment adsorbents and were investigated for their cross-reaction with the other two fragments. Specific antibodies against each subdomain were found to cross-react with the other two subdomains. The degree of cross-reaction increased remarkably with the duration of immunization and reached as high as 100% for the cross-reaction between subdomains 3 and 6. The results are consistent with an unusual functional equivalence of antigenic sites within the same protein molecule. This functional equivalence of the antigenic sites of albumin, which we had previously proposed, improves with time after immunization.

Immunochemistry of serum albumin—VIII: The antigenic reactivity of the third domain of bovine serum albumin resides in the last sub-domain. a dynamic examination of the change of antibody affinity and specificity☆

Abstract In a preceding paper, immunochemical and conformational studies on chemical derivatives of fragment 377–571 of bovine serum albumin (BSA) suggested that the immunochemical reactivity of this fragment (the third domain of the albumin molecule) resided entirely in the last sub-domain. In the present work, the immunochemical reactivities of fragments 377–571 and 504–581 with serial antisera to BSA were studied in detail. Antisera against BSA were raised in two rabbits and serial bleedings were obtained at different periods after the first immunization. The serial antisera were not mixed but were kept and studied separately. Immunoadsorbent titration studies showed that the plateau values of 125 I-antibody binding by the immunoadsorbents of the two fragments changed with time after the initial immunization but were, for a given antiserum, identical in all serial antisera from 15 days up to 398 days. However, in very early antisera (7 days) the larger fragment 377–571 possessed a higher immunochemical reactivity than the small fragment 504–581. The inhibitory activities of the two fragments towards the binding of 125 -albumin and 125 I-fragment 377–571 with serial antisera to BSA in a Farr assay were compared. The two fragments exhibited comparable inhibitory activities and, in addition, fragment 504–581 was able to inhibit completely the binding of 125 I-fragment 377–571. Acid dissociation studies of complexes of serial 125 I-antibodies on immunoadsorbents revealed that the affinity of the antibodies increased while their heterogeneity decreased with time after immunization. The results with serial antisera from each of the two rabbits show that although in very early (7 days) antisera, the larger fragment appears to carry some additional antigenic sites, the shared antigenic sites become completely immunodominant relatively early (15 days) after the first immunization.

Antibodies to synthetic antibody-combining sites. Antibodies against a surface-simulation peptide with antibody-combining activity toward lysozyme antigenic site 3 react with lysozyme antibodies.

Recently, we reported the synthesis and immunochemistry of two peptides designed, by complementarity and surface-simulation synthesis, to mimic antibody-combining sites against two antigenic sites of lysozyme. In the present work antibodies were raised against one of these peptides, which is complementary to antigenic site 3 of lysozyme, to determine whether these antibodies will react with anti-lysozyme antibodies. Radioiodinated antipeptide antibodies were bound by immunoadsorbents of the immune IgG from two goats anti-lysozyme antisera but not by adsorbents of myoglobin, non-immune goat IgG or immune IgG of antisera against cytochrome c. The binding of anti-peptide antibodies to adsorbents of anti-lysozyme antibodies was fully inhibited by free lysozyme but not by bovine serum albumin, human hemoglobin A, horse cytochrome c or bovine ribonuclease A. Thus, antisera against an antibody-combining site can be raised by immunizing with a peptide which probably does not exist in the antibody but is designed by surface-simulation synthesis to mimic an antibody-combining site.

Immune recognition of serum albumin. XVI. role of adjuvant in the autoimmune response to mouse serum albumin

Abstract Recently, we reported an autoreactivity with rabbit serum albumin of rabbit antisera against bovine serum albumin (BSA) or human serum albumin. Also immunization of rabbits, each with its own serum albumin in complete Freunds adjuvant (CFA), gave without exception an autoantibody response to rabbit serum albumin. The effect of adjuvant in the autoimmune response was proposed and the present work has investigated this effect. Mice immunized with BSA emulsified in CFA gave a high antibody response to BSA which exhibited considerable autoreactivity with mouse serum albumin (MSA). When BSA was injected in buffered saline (PBS) at one site and CFA was injected at a different site but on the same side of the animal, a lower antibody response (to BSA) was observed and the antibodies exhibited a lower autoreactivity with MSA than when BSA-CFA emulsion was employed. No autoreactivity was observed when BSA in PBS and CFA were injected on two different sides of the animal or when BSA in PBS was injected without adjuvant. Injection of CFA alone generated no antibody response. Similar experiments were carried out in which MSA was injected in mice. Autoantibodies were obtained when MSA in CFA emulsion was injected. A marginal autoantibody response was detected when MSA in PBS and CFA were injected at different sites on the same side of the animal. All other protocols, including emulsion in incomplete Freunds adjuvant, failed to stimulate an autoantibody response. The results indicate that CFA plays a critical role in the autoimmune response and that adjuvant and antigen must act on the same lymph nodes to mount a fruitful autoimmune response.