Multiple overlapping epitopes in the three antigenic regions of horse cytochrome c1.
Abstract: To gain a better understanding of the diversity of epitopes on a protein, the specificities of 103 monoclonal antibodies to a model antigen, horse cytochrome c(cyt c), were analyzed. The antibodies were generated in in vitro monoclonal, secondary antibody responses against horse cyt c coupled to hemocyanin in splenic fragment cultures. For this assay, horse cyt c-primed murine B lymphocytes were transferred to irradiated, hemocyanin-primed recipients. A panel of seven mammalian cyts c differing at one to six residues out of 104 and cyanogen bromide-cleaved fragments of horse cyt c containing residues 1-65, 1-80, and 66-104 was used to examine the specificities of the antibodies. Twenty-two distinct reactivity patterns were observed, even though the majority of the monoclonal antibodies were found to bind in the three previously identified antigenic regions of the molecule about residues 44-47, 60-62, and 89-92. The results indicate that each of the three antigenic regions consists of multiple overlapping epitopes. Few of the antibodies directed to any given antigenic region bound polypeptide fragments inclusive of the epitope sequences, demonstrating that some antibodies were more conformationally dependent than others. Only 13% of the antibodies bound to cyanogen bromide-cleaved polypeptide fragments that together encompassed the entire length of the protein. Considering the large number of antibodies analyzed and the reoccurrence of 13 of the 22 clonotypes in different lymphocyte donors, it is likely that the antibody specificities tabulated herein approach yet do not completely enumerate the total inventory of the horse cyt c-specific B cell repertoire. The remarkable diversity for epitope recognition within antigenic regions observed here is likely to pertain to protein antigens in general, and strongly supports the widely held notion that the entire surface of a protein is potentially antigenic. The restriction of the epitopes of horse cyt c to three antigenic regions where the amino acid sequences of the mammalian cyts c differ probably results from tolerance of the mice to their own cyt c.
Publication Date: 1987-01-01 PubMed ID: 2431055
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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The research is about studying the diversity of epitopes (specific area on an antigen that an antibody recognizes and binds to) on a protein by using horse cytochrome c as a model. The researchers found that each of the three antigenic regions of the horse cytochrome c consists of multiple overlapping epitopes and examined the reactivity of 103 different monoclonal antibodies, generated in vitro, to these regions.
Overview of the Research
- The goal of this study was to explore the diversity of epitopes on a protein using horse cytochrome c as a model. This involved generating monoclonal antibodies in vitro and investigating their specificities.
- The researchers used a wide variety of monoclonal antibodies, totaling 103, which were produced in monoclonal, secondary antibody responses against horse cytochrome c.
- The horse cytochrome c was coupled with hemocyanin, a blue protein found in the blood of some marine mollusks and arthropods, in splenic fragment cultures to help trigger reactions.
- Horse cytochrome c-primed murine B lymphocytes were utilized in the process and were moved to irradiated, hemocyanin-primed recipients to conduct the assay.
Findings and Analysis
- With the help of seven mammalian cytochromes c and cyanogen bromide-cleaved fragments of horse cytochrome c, the researchers were able to examine the specificities of the antibodies.
- Though many of the monoclonal antibodies bound to the three known antigenic regions on the molecule, they observed 22 distinct reactivity patterns, indicating that each region is made up of numerous overlapping epitopes.
- They found that a small proportion of the antibodies, only 13%, bound to cyanogen bromide-cleaved polypeptide fragments spanning the entire length of the protein. The majority of the antibodies showed a dependency on the conformation, or structure, of the epitopes.
- The results suggest that the diversity of epitope recognition within antigenic regions is likely to extend to protein antigens in general, implying that the entire surface of a protein could potentially be antigenic.
Conclusion
- Based on the results, the researchers concluded that the three antigenic regions where the amino acid sequences of the mammalian cytochromes c differ were likely a result of the mice’s tolerance to their own cytochrome c.
- Despite analyzing a large number of antibodies, the researchers acknowledge that the specificities recorded in the study may not entirely enumerate the total inventory of the horse cytochrome c-specific B cell repertoire.
Cite This Article
APA
Jemmerson R.
(1987).
Multiple overlapping epitopes in the three antigenic regions of horse cytochrome c1.
J Immunol, 138(1), 213-219.
Publication
Researcher Affiliations
MeSH Terms
- Amino Acid Sequence
- Animals
- Antibodies, Monoclonal / immunology
- B-Lymphocytes / immunology
- Cytochrome c Group / analogs & derivatives
- Cytochromes c1 / immunology
- Epitopes
- Horses
- Immunologic Memory
- Peptide Fragments / immunology
- Receptors, Antigen, B-Cell / immunology
- Species Specificity
Grant Funding
- AI-23803 / NIAID NIH HHS
Citations
This article has been cited 6 times.- Schmohl JU, Todhunter D, Oh S, Vallera DA. Mutagenic Deimmunization of Diphtheria Toxin for Use in Biologic Drug Development.. Toxins (Basel) 2015 Oct 10;7(10):4067-82.
- Nagata S, Pastan I. Removal of B cell epitopes as a practical approach for reducing the immunogenicity of foreign protein-based therapeutics.. Adv Drug Deliv Rev 2009 Sep 30;61(11):977-85.
- Philibert P, Stoessel A, Wang W, Sibler AP, Bec N, Larroque C, Saven JG, Courtête J, Weiss E, Martineau P. A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm.. BMC Biotechnol 2007 Nov 22;7:81.
- Puttick AH, Williamson EA, Merry AH, Kumpel BM, Thompson KM, Jones VE. Reaction of rheumatoid factors with IgG3 monoclonal anti-Rh(D) antibodies: more frequent reactivity to a monoclonal antibody of the Gm allotype G3m(5) in rheumatoid patients negative for G3m(5).. Ann Rheum Dis 1988 Nov;47(11):898-905.
- Fink JM, Kalos M, Zissler JF. Isolation of cell surface antigen mutants of Myxococcus xanthus by use of monoclonal antibodies.. J Bacteriol 1989 Apr;171(4):2033-41.
- Jemmerson R. Antigenicity and native structure of globular proteins: low frequency of peptide reactive antibodies.. Proc Natl Acad Sci U S A 1987 Dec;84(24):9180-4.
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