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Yari F, Gorzin F. A Dimerized Synthetic RhD Peptide for Rabbit Immunization: Comparison With the Antigen from Human Red Blood Cell Membranes. bloodj 2026; 23 (1) :45-52
URL: http://bloodjournal.ir/article-1-1623-en.html
A Dimerized Synthetic RhD Peptide for Rabbit Immunization: Comparison With the Antigen from Human Red Blood Cell Membranes
Fatemeh Yari * , Fatemeh Gorzin
Keywords: Red Blood Cells, Rh Factor, Peptides, Immunization, Rabbits
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   References:
  1. Avent ND, Reid ME. The Rh blood group system: a review. Blood. 2000; 95(2): 375-87. [DOI:10.1182/blood.V95.2.375] [PMID]
  2. Westhoff CM. The structure and function of the Rh antigen complex. Semin Hematol. 2007; 44(1): 42-50. [DOI:10.1053/j.seminhematol.2006.09.010] [PMID] []
  3. Nadarajan VS. Serological analysis of Rh antigens: how far can we go? Annals of Blood. 2023; 8. [DOI:10.21037/aob-23-30]
  4. 4. Li HY, Guo K. Blood Group Testing. Front Med (Lausanne). 2022; 9: 827619. [DOI:10.3389/fmed.2022.827619] [PMID] []
  5. Wu H, Li R, Wei H, Zhu W, Xing Y. Clinical Characteristics and Prognosis of Hemolytic Disease of the Newborn Caused by Irregular Antibodies: A 13-Year Retrospective Analysis. Children (Basel). 2024;11(12): 1409. [DOI:10.3390/children11121409] [PMID] []
  6. Grant GA. Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins. Curr Protoc Immunol. 2003; 55(1): 9.2: 1-9. [DOI:10.1002/0471142735.im0902s55] [PMID]
  7. Tamura T, Tomimatsu K, Katakura Y, Yamashita M, Matsumoto SE, Aiba Y, et al. Anti-peptide antibody production elicited by in vitro immunization of human peripheral blood mononuclear cells. Biosci Biotechnol Biochem. 2007; 71(12): 2871-5. [DOI:10.1271/bbb.60460] [PMID]
  8. Lee BS, Huang JS, Jayathilaka LP, Lee J, Gupta S. Antibody Production with Synthetic Peptides. Methods Mol Biol. 2016; 1474: 25-47. [DOI:10.1007/978-1-4939-6352-2_2] [PMID]
  9. Millard AL, Ittelet D, Schooneman F, Bernard J. Dendritic cell KLH loading requirements for efficient CD4+ T-cell priming and help to peptide-specific cytotoxic T-cell response, in view of potential use  in cancer vaccines. Vaccine. 2003; 21(9-10): 869-76. [DOI:10.1016/S0264-410X(02)00534-0] [PMID]
  1. Cochet S, Blancher A, Roubinet F , Hattab C , Cartron JP, Bertrand O. Immunopurification of the blood group RhD protein from human erythrocyte membranes. J Chromatogr B Biomed Sci Appl. 1999 10; 735(2):207-17. [DOI:10.1016/S0378-4347(99)00424-7] [PMID]
  2. Rezaeeyan H , Yari F, Milani S. The Blood Group Rhc Protein from Human Erythrocyte Membranes as an Immunogen for Producing Antibodies in Mice. Iran J Ped Hematol Oncol. 2024; 14( 1): 44-52. [DOI:10.18502/ijpho.v14i1.14663]
  3. Greg T. Hermanson, editor: Bioconjugation. 3rd ed. UK: Pierce Biotechnology, Thermo Fisher Scientific, Rockford, IL; 2013: Chapter 13
  4. Johnstone A and Thorpe R, editors: Immunochemistry in Practice. 3rd ed. London: Blackwell Science; 1996: Chapter 2
  5. Walker RY, Andrew S, Kumpel BM, Austin EB. Murine monoclonal antibodies reactive with a human monoclonal anti-RhD antibody (BRAD-5). Transfus Med. 2000; 10(3): 225-31. [DOI:10.1046/j.1365-3148.2000.00258.x] [PMID]
  6. Miescher S, Zahn-Zabal M, De Jesus M, Moudry R, Fisch I, Vogel M, et al. CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display. Br J Haematol. 2000;111(1): 157-66. https://doi.org/10.1111/j.1365-2141.2000.02322.x [DOI:10.1046/j.1365-2141.2000.02322.x]
  7. Hermand P, Mouro I, Huet M, Bloy C, Suyama K, Goldstein J, et al. Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides. Blood. 1993 ;82(2): 669-76. https://doi.org/10.1182/blood.V82.2.669.bloodjournal822669 [DOI:10.1182/blood.V82.2.669.669]





A Dimerized Synthetic RhD Peptide for Rabbit Immunization: Comparison With the Antigen from Human Red
Blood Cell Membranes

Fatemeh Yari1      , Fatemeh Gorzin1



1Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

Received: 2026/04/13
Accepted: 2026/05/13
 





     http://dx.doi.org/10.61186/bloodj.21.4.333

    



Citation:
Yari F, Gorzin F. A Dimerized Synthetic RhD Peptide for Rabbit Immunization: Comparison With the Antigen from Human Red Blood Cell Membranes. J Iran Blood Transfus. 2026: 23 (1): 45-52
    



Correspondence: Yari F., Professor of Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine.
P.O.Box: 14665-1157, Tehran, Iran.
Tel: (+9821) 82052237
E-mail: f.yari@
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tmi.ac.ir  		 A B S T R A C T
Background and Objectives
The Rh proteins play a crucial role in blood transfusion. Among the Rh blood group antigens, the D antigen is notable for its high immunogenicity. The aim of this study was to produce specific antibodies against the  RhD antigen in rabbits by utilizing a complete and dimeric antigen derived from a synthetic peptide specific to the D antigen. Ultimately, the potential of these antibodies to agglutinate human red blood cells was investigated.
Materials and Methods
In this experimental study, antibodies against RhD were generated in rabbits. Rabbits 1 and 2 were immunized with the complete RhD antigen, which was purified from the membranes of red blood cells by affinity chromatography. Concurrently, Rabbit 3, was immunized with an RhD antigen dipeptide, which was prepared from a selected peptide of RhD using the O-PDM method. IgG molecules were purified from the antisera produced in rabbits. The characteristics of both the purified RhD antigen and the antisera were evaluated using the ELISA method. Additionally, the functional activity of antisera was assessed through the agglutination of human red blood cells.
Results
The characteristic of the purified RhD antigen was demonstrated by ELISA assay using commercial anti-RhD antibody. Antibody formation in the sera of rabbits was also confirmed using the ELISA method. The isolated IgG polyclonal rabbit antibodies against RhD showed agglutination of O+ red blood cells in the presence of a secondary antibody (n=3). Subsequently, the specificity of the produced antisera was similar between rabbits immunized with the purified antigen and  those immunized with the dimerized peptide. The antibodies were only able to agglutinate red blood cells through a two-step method. The synthetic peptide related to the amino terminus of RhD antigen, in its dimeric form, exhibited immunogenic characteristic comparable to those of the complete antigen.
Conclusions 
These findings indicate that the appropriate dimeric form of a peptide can serve as a substitute for the conjugated peptide and a carrier protein for achieving successful immunization.
Key words: Red Blood Cells, Rh Factor, Peptides, Immunization, Rabbits
Copyright © 2025 Journal of Iranian Blood Transfusion, Published by Blood Transfusion Research Center.
This work is licensed under a Creative Common Attribution-Non Commercial 4.0 International license.


 
Type of Study: Research | Subject: Imunology
References
1. Avent ND, Reid ME. The Rh blood group system: a review. Blood. 2000; 95(2): 375-87. [DOI:10.1182/blood.V95.2.375] [PMID]
2. Westhoff CM. The structure and function of the Rh antigen complex. Semin Hematol. 2007; 44(1): 42-50. [DOI:10.1053/j.seminhematol.2006.09.010] [PMID] []
3. Nadarajan VS. Serological analysis of Rh antigens: how far can we go? Annals of Blood. 2023; 8. [DOI:10.21037/aob-23-30]
4. 4. Li HY, Guo K. Blood Group Testing. Front Med (Lausanne). 2022; 9: 827619. [DOI:10.3389/fmed.2022.827619] [PMID] []
5. Wu H, Li R, Wei H, Zhu W, Xing Y. Clinical Characteristics and Prognosis of Hemolytic Disease of the Newborn Caused by Irregular Antibodies: A 13-Year Retrospective Analysis. Children (Basel). 2024;11(12): 1409. [DOI:10.3390/children11121409] [PMID] []
6. Grant GA. Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins. Curr Protoc Immunol. 2003; 55(1): 9.2: 1-9. [DOI:10.1002/0471142735.im0902s55] [PMID]
7. Tamura T, Tomimatsu K, Katakura Y, Yamashita M, Matsumoto SE, Aiba Y, et al. Anti-peptide antibody production elicited by in vitro immunization of human peripheral blood mononuclear cells. Biosci Biotechnol Biochem. 2007; 71(12): 2871-5. [DOI:10.1271/bbb.60460] [PMID]
8. Lee BS, Huang JS, Jayathilaka LP, Lee J, Gupta S. Antibody Production with Synthetic Peptides. Methods Mol Biol. 2016; 1474: 25-47. [DOI:10.1007/978-1-4939-6352-2_2] [PMID]
9. Millard AL, Ittelet D, Schooneman F, Bernard J. Dendritic cell KLH loading requirements for efficient CD4+ T-cell priming and help to peptide-specific cytotoxic T-cell response, in view of potential use in cancer vaccines. Vaccine. 2003; 21(9-10): 869-76. [DOI:10.1016/S0264-410X(02)00534-0] [PMID]
10. Cochet S, Blancher A, Roubinet F , Hattab C , Cartron JP, Bertrand O. Immunopurification of the blood group RhD protein from human erythrocyte membranes. J Chromatogr B Biomed Sci Appl. 1999 10; 735(2):207-17. [DOI:10.1016/S0378-4347(99)00424-7] [PMID]
11. Rezaeeyan H , Yari F, Milani S. The Blood Group Rhc Protein from Human Erythrocyte Membranes as an Immunogen for Producing Antibodies in Mice. Iran J Ped Hematol Oncol. 2024; 14( 1): 44-52. [DOI:10.18502/ijpho.v14i1.14663]
12. Greg T. Hermanson, editor: Bioconjugation. 3rd ed. UK: Pierce Biotechnology, Thermo Fisher Scientific, Rockford, IL; 2013: Chapter 13
13. Johnstone A and Thorpe R, editors: Immunochemistry in Practice. 3rd ed. London: Blackwell Science; 1996: Chapter 2
14. Walker RY, Andrew S, Kumpel BM, Austin EB. Murine monoclonal antibodies reactive with a human monoclonal anti-RhD antibody (BRAD-5). Transfus Med. 2000; 10(3): 225-31. [DOI:10.1046/j.1365-3148.2000.00258.x] [PMID]
15. Miescher S, Zahn-Zabal M, De Jesus M, Moudry R, Fisch I, Vogel M, et al. CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display. Br J Haematol. 2000;111(1): 157-66. https://doi.org/10.1111/j.1365-2141.2000.02322.x [DOI:10.1046/j.1365-2141.2000.02322.x]
16. Hermand P, Mouro I, Huet M, Bloy C, Suyama K, Goldstein J, et al. Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides. Blood. 1993 ;82(2): 669-76. https://doi.org/10.1182/blood.V82.2.669.bloodjournal822669 [DOI:10.1182/blood.V82.2.669.669]
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