PubMed Health⌕ Search

Biomedical subjects

M Z Atassi

Publications and source records attributed to M Z Atassi.

At least 145 records · Page 8Linked to original sources
PubMed1983

Antibodies with preselected specificities to protein regions evoked by immunization with free synthetic peptides: dose response to myoglobin antigenic sites reveal an optimum dose for each antigenic site.

Previously, we have shown by radioimmune antibody binding studies that immunization with each of the five synthetic antigenic sites of sperm-whale myoglobin (Mb), in their free form (i.e. not coupled to a carrier), resulted in the formation of antibodies that bound specifically to Mb and exclusively to the peptide that was used in immunization. In the present series of studies we have immunized separate groups of Balb/cByJ mice with different amounts of each of the free synthetic antigenic sites so as to determine the optimum immunizing dose for eliciting serum antibodies that bind specifically to Mb. The synthetic peptides corresponded to: Site 1 (peptide 15-22), Site 2 (peptide 56-62), Site 3 (peptide 94-100), Site 4 (peptide 113-120, and Site 5 (peptide 145-151). The dose range of each of the antigenic sites used in immunization was from 6 micrograms to 100 micrograms. Radioimmune antibody binding studies indicated that there is an optimal immunizing dose for each of the five antigenic sites which was smaller than anticipated. The significance of these findings is discussed.

Animals↗
PubMed1983

Production of monoclonal antibodies to surface regions that are non-immunogenic in a protein using free synthetic peptide as immunogens: demonstration with sperm-whale myoglobin.

Two synthetic peptides corresponding to surface regions of sperm whale myoglobin that are not antigenic in the native molecule were used in their free form (i.e. not coupled to a carrier) to immunize separate groups of Balb/cByJ mice. The synthetic peptides corresponded to regions 1-6 and 121-127. Serum samples obtained from each group of mice contained antibodies that bound specifically to myoglobin and exclusively to the immunizing peptide. Monoclonal antibodies to each of the two surface regions were subsequently obtained by hybridizing Fa/O mouse myeloma cells with spleen cells derived from each group of mice. These monoclonal antibodies were IgM (kappa). They expressed the same isotype as the antigen specific serum antibodies produced by the mice whose spleen cells were used for hybridization. Solid phase radioimmunoassay studies also indicated that each monoclonal antibody, like the immune serum of the parent animals, bound specifically to myoglobin and exclusively to the synthetic peptide used as an immunogen. These results suggested that the hybridoma antibodies expressed submolecular binding specificities that were the result of peptide immunization rather than hybrid selection and that monoclonal antibodies with preselected submolecular binding specificities to non-antigenic surface regions in a protein molecule can be produced by the techniques of somatic cell hybridization when the corresponding free synthetic peptides are used as immunogens.

Animals↗
PubMed1982

Immune recognition of serum albumin.15. Localization by synthesis of antigenic site 4 of bovine serum albumin to the region around the disulfide bond 166-175.

Recently, we have localized and confirmed by synthesis the regions within which reside five major antigenic sites of bovine serum albumin and proposed that the remaining sixth major antigenic site (antigenic site 4) was localized within subdomain 3 (fragment 115-184) of albumin. In the present work, antigenic site 4 was localized to fall around the disulfide bond 166-175 by synthesis and immunochemical reactivity of the region 162-179. The synthetic 18-residue peptide was shown to bind substantial amounts of antibodies from early (38-day) and late (398-day) anti-albumin antisera from two rabbits. As much as half the total anti-albumin antibodies could be bound by the peptide from the late antisera. It was concluded that antigenic site 4 resides within, but may not include all of, the region 162-179 of albumin.

Amino Acid Sequence↗
PubMed1982

Structurally inherent antigenic sites. Localization of the antigenic sites of the alpha-chain of human haemoglobin in three host species by a comprehensive synthetic approach.

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.

Amino Acid Sequence↗
PubMed1982

Binding with lysozyme of antibodies against surface-simulation peptides representing the lysozyme antigenic sites.

Previously it had been shown that native lysozyme has three discontinuous antigenic sites (comprising spatially adjacent residues that may be distant in sequence) that were mimicked by surface-simulation synthetic peptides that had the capacity to bind the bulk (97-99%) of the antibody response against native lysozyme. In the present work these three surface-simulation synthetic peptides were coupled to succinoylated bovine serum albumin, and the conjugates were injected into rabbits. Antibodies against each peptide reacted, as expected, only with that peptide, but it was also found that the antibodies could bind with lysozyme, and the complete specificity of this binding was rigorously established. The advantages of these findings in conformational and immunological investigations are outlined.

Antibodies↗
PubMed1982

Dissection of the molecular parameters for T-cell recognition of a myoglobin antigenic site.

Previous studies from this laboratory have resulted in the determination of the antigenic structure of sperm-whale myoglobin (Mb). In the present work, we have investigated the fine specificity requirements for T-cell recognition of one of the Mb antigenic sites (antigenic site 5). The antigenic site (peptide 145-153) and seven progressively longer peptides, increasing in length stepwise by two residues at a time up to 22 residues in length (peptide 132-153), were synthesized. In addition, four truncated peptides were synthesized with intentional deletions at Tyr-151 and Ala-144. The T-cell recognition of these purified synthetic peptides was examined here in detail in three strains of mice (BALB/cByJ, B10.D2/n and SJL/J). Mb-primed mice afforded T-cells which proliferated to smaller peptides (two or four residues longer than the site; i.e. peptides 145-153 and 143-153) and more so to the longer peptides 135-153 and 132-153 and to Mb. No response was obtained to the truncated peptides, thus underscoring the fine specificity of T-cells. No response was obtained also to intermediate-sized peptides. The latter result, due to an unfavorable mode of folding, suggested a conformational dependency in T-lymphocyte recognition and was confirmed by additional studies employing peptide priming. Thus priming with either the 11- or 22-residue peptides afforded T-cells which proliferated to both of these two peptides, but not to intermediate-sized peptides. More importantly, peptide-primed cells did not proliferate on challenge in vitro with Mb. This indicated that the in vivo recognition (priming) was to the unfolded peptide and these cells will not therefore recognize native Mb in vitro. Tolerization of neonatal mice with native Mb, which was total for the native protein at the T-cell level, had no effect on in vivo recognition (priming) and in vitro proliferation due to the unfolded peptide. Conversely, tolerization by any unfolded peptide had little or no effect on in vivo recognition of, and proliferation to, native Mb. It was concluded that T-cell recognition of native proteins (or at least of Mb) is dependent on protein conformation. A peptide may not cause proliferation of native protein-primed T-cells even though it contains an antigenic site. Finally, macrophage presentation of protein antigens is not related to processing via fragmentation of antigen by macrophage.

Animals↗
PubMed1982

Genetic control and intersite influences on the immune response to sperm whale myoglobin.

Determination of the precise antigenic structure of sperm-whale myoglobin (Mb) has enabled us to focus our attention on the molecular and cellular factors that control and regulate the immune responses to the protein antigen. Our studies have shown that the immune responses to sperm-whale Mb are controlled by genes in the I region of the major histocompatibility complex (H-2) of mice. More importantly, the responses to the synthetic antigenic sites are each under separate genetic control. The recognition of the antigenic sites by antibodies is independent of the immunized species and of the time the antisera are obtained after the initial immunization (from nine days up to a year). The same sites are recognized by antisera raised in rabbit, goat, pig, cat, chicken and outbred and inbred mice. The same sites recognized by mouse B-cells are also recognized by mouse T-cells. No meaningful genetic control of antibody affinity was observed. Autoimmune antibody and T-lymphocyte proliferative responses were readily generated by immunizing an animal with self-Mb. With mouse Mb, the autoimmune T-lymphocyte response was under genetic control and mapped with the I-A and the H-2D end of the H-2 gene complex. In other recent studies we have shown, using several Mb variants, that the binding capacity of an antigenic site is fully accounted for by substitutions in the antigenic sites (actual contact residues) and in residues close (within 7.A) to the sites (i.e. environmental residues). The overall response to Mb is regulated by inter-site influences which can either be of a cooperative (help) nature or of a suppressive nature. Finally, genetic control of the responses to individual antigenic sites on a protein is not only determined by the genetic constitution of the host but also by the chemical properties of the individual sites. The H-2 subregions mapping the responses to given antigenic sites can also recognize other sites, which were previously unrecognizable in a homologous protein, if the chemical properties of these sites are suitably altered.

Animals↗
PubMed1982

Production of monoclonal antibodies with pre-selected submolecular binding specificities to protein determinants: demonstration with sperm whale myoglobin.

Two monoclonal antibodies with pre-selected submolecular binding specificities to sperm whale myoglobin (Mb) were obtained by hybridizing Fa/0 mouse myeloma cells with spleen cells derived from mice which had been immunized with free (not coupled to any carrier) Mb synthetic peptides 132-153 or 145-151 (antigenic site 5). Both monoclonal antibodies were IgG1 (k). Their homogeneity was confirmed by analytical isoelectric focusing electrophoresis. According to competitive inhibition studies in which Mb, the five synthetic antigenic sites of Mb, Mb peptide 132-153, bovine serum albumin (BSA), and lysozyme were used as inhibitors, the binding specificities of both monoclonal antibodies were restricted to determinants present in the peptides used for immunizations. The results of direct binding studies confirmed this conclusion and suggested that monoclonal antibodies with pre-selected submolecular binding specificities can be readily obtained by the techniques of somatic cell hybridization when the corresponding free synthetic determinants are used as immunogens.

Animals↗
PubMed1982

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.

Animals↗
PubMed1982

Genetic control of the immune response to haemoglobin. IV. Ly-1+ T-cells and appropriate non-H-2 genes are required for in vitro responses to alpha- and beta-subunits of human adult haemoglobin.

Experiments were conducted to determine if non-H2 gene effects could be demonstrated in mice which had been primed to either the alpha-subunit or beta-subunit of human haemoglobin. It was found that C3H.SW (H-2b) and Balb/c (H-2d) mice are low responder mice to alpha-chain of a haemoglobin when compared to H-2 identical B10 (H-2b) and B10.D2(H-2d) mice. B120.S and A.SW (both H-2s) are responsive to beta-chain challenge while Balb/c mice are low responders in contrast to high responder B10.D2 mice. Ly-1+ cells were demonstrated to be required (by cell depletion experiments) for an in vitro T-cell proliferative response to either subunit. In these experiments, Ly-2+ cells were not of crucial importance.

Animals↗
PubMed1982

Genetic control of the immune response to myoglobin. IX. Overcoming genetic control of antibody response to antigenic sites by increasing the dose of antigen used in immunization.

Previously, it was reported that the immune response to myoglobin (Mg) was under genetic control, with the response to each site being under separate Ir-gene control. Here we have investigated the effect of antigen dose on the control of the antibody response to the five antigenic sites of sperm-whale Mb to determine whether or not the overcoming of genetic control by antigen dose has a uniform effect on all five antigenic sites. The antibody response to sperm whale myoglobin (Mb) and its five antigenic was measured in the following inbred strains of mice, C57BL/6J, AKR and SWR/J. These strains of mice are low responders to Mb following immunization with 50 micrograms, responding only to site 4. After immunization with 200 micrograms Mb: C57BL/6J mice are high responders to Mb and respond to antigenic sites 1, 3, 4 and 5; AKR mice are high responders to Mb and respond to antigenic sites 1 and 4; SWR/J mice are high responders to Mb and respond to all five antigenic sites. It was concluded that the genetic control of the immune response to Mb and its synthetic antigenic sites is dependent on antigen dose. Also, these studies have enabled us for the first time to separate the response to site 1 from the response to site 2 and thus have conclusively established that sites 1 and 2 are controlled by separate Ir-genes.

Amino Acid Sequence↗
PubMed1982

Antibodies to sperm-whale myoglobin evoked by free synthetic peptides of an antigenic site.

Previous studies from this laboratory have resulted in the determination of the entire antigenic structure of myoglobin (Mb). The present work was carried out to investigate the antigenicity of the synthetic antigenic sites of Mb in their free form (i.e. without coupling to a carrier) and the effect of peptide size on antigenicity. Site 5 (peptide 145-151) and the longer peptides 145-153, 143-153 and 132-153, each of which carries within it the region of site 5, were synthesized. Immunization of three different mouse strains with each of these peptides, in their free form, resulted in the formation of antibodies that bound specifically to intact Mb. The advantages and significance of these findings are discussed.

Amino Acid Sequence↗
PubMed1981

T cell clones reactive with sperm whale myoglobin. Isolation of clones with specificity for individual determinants on myoglobin.

We have been able to isolate clones of sperm whale muscle myoglobin (Mb)-reactive T cells from (C57BL/6 x A/J)F1 [(B6A)F1] mice. Four types of clones were isolated, distinguished by their patterns of recognition of Mb cyanogen bromide (CNBr) fragments and antigen presenting cell (APC) requirements. Individual T cell clones proliferated in response to one of three CNBr fragments of Mb. Dose-response curves of all clones were identical for native Mb and the appropriate fragment. T cell clones reactive to fragment 1-55 did not proliferate in response to peptide 15-22 (a peptide that binds to serum antibody directed against 1-55). These data support previous findings suggesting differences between antigen recognition by T and B cells, i.e., T cells may not recognize antigen in its native conformation and/or T and B cells may recognize distinct epitopes on the same antigen. Using T cell clones to analyze genetic control of responsiveness to Mb, we found that certain (B6A)F1 T cells recognize Mb presented by low responder strain APC. Thus, genetically determined low responsiveness in this case is probably not due to failure of APC function. We also found that responsiveness to certain Mb epitopes mapped to the I-A subregion whereas others mapped, via gene complementation, to the I-A and I-E subregions. We found no examples of responsiveness mapping to the I-C subregion and suggest an alternative explanation for previous reports mapping genetic control of responsiveness to certain Mb determinants to I-C.

Animals↗
PubMed1981

High yield coupling of peptides to protein carriers.

Coupling of peptides to protein carriers has been achieved at a high level of peptide incorporation. Bovine serum albumin was used as the model carrier protein and several peptides were tested in the coupling reaction. The peptides were coupled to succinylated bovine serum albumin after conversion of its carboxyl side chains to the reactive p-nitrophenyl ester group by reaction with p-nitrophenol/N,N'-dicyclohexylcarbodiimide in anhydrous dimethylformamide. The reaction afforded succinyl-albumin-peptide conjugates that had high level of peptide incorporation (18-35 mol peptide/mol albumin). In addition to the high level of peptide coupling, the reaction avoids the production of polymeric forms of peptide, protein or conjugate.

Carrier Proteins↗
PubMed1981

Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding site on the alpha-chain of human haemoglobin.

A synthetic approach was employed to identify the haptoglobin-binding site on the alpha-chain of human haemoglobin. This approach cosists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen peptides were synthesized (alpha 1-15, alpha 11-25, alpha 21-35, alpha 31-45, alpha 41-55, alpha 51-65, alpha 61-75, alpha 71-85, alpha 81-95, alpha 91-105, alpha 101-115, alpha 111-125, alpha 121-135 and alpha 131-141), and their ability bind human haptoglobin was studied, Only peptide alpha 121-135 bound haptoglobin significantly. On this basis we conclude that the haptoglobin-binding site on the alpha-chain of haemoglobin resides within, but does not necessarily encompass all of, the region alpha 121-135.

Binding Sites↗