Volume 34, Issue 5 (August 2023)
Studies in Medical Sciences 2023, 34(5): 247-258 |
Back to browse issues page
Research code: 20195
Ethics code: IR.SUMS.MED.REC.1399.193
Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Mehdipour F, Mirshahi A, Hoseini A, Mirshahi M, Ghaderi A. PRODUCTION AND PURIFICATION OF RABBIT ANTI-CD166 POLYCLONAL ANTIBODY. Studies in Medical Sciences 2023; 34 (5) :247-258
URL: http://umj.umsu.ac.ir/article-1-6032-en.html
URL: http://umj.umsu.ac.ir/article-1-6032-en.html
Professor of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran (Corresponding Author) , ghaderia@sums.ac.ir
Abstract: (1015 Views)
Background & Aims: CD166 is an Activated Leukocyte Cell Adhesion Molecule (ALCAM) which acts as a cell-to-cell adhesion molecule. CD166 has an important role in the growth, survival and invasion of the tumor cells. The aim of the present study was to produce polyclonal antibodies against CD166.
Materials & Methods: The selected peptide was synthesized from the CD166 molecule and after conjugation with KLH protein, in an emulsion with Freund's complete adjuvant, it was injected subcutaneously to two female New Zealand rabbits several times. Then the antibodies in rabbit serum were purified by FPLC method and their amounts were determined by indirect ELISA method. By CNBr gel purification method, specific antibodies against CD166 peptide bound to KLH were isolated, and the concentration of produced antibody against peptide or KLH-peptide was determined by ELISA method.
Results: After repeated injection of CD166 peptide bound to KLH, antibody against CD166 peptide was produced, but it had a low titer. Most of the antibody produced was directed against the KLH-peptide binding site. Also, the tendency of produced antibodies to bind to KLH-peptide was higher compared to the peptide alone.
Conclusion: The produced antibodies were mostly against the antigenic index of KLH peptide. The synthetic and small size of the injected peptide can be the reason for the low titer and affinity of the antibody to it.
Materials & Methods: The selected peptide was synthesized from the CD166 molecule and after conjugation with KLH protein, in an emulsion with Freund's complete adjuvant, it was injected subcutaneously to two female New Zealand rabbits several times. Then the antibodies in rabbit serum were purified by FPLC method and their amounts were determined by indirect ELISA method. By CNBr gel purification method, specific antibodies against CD166 peptide bound to KLH were isolated, and the concentration of produced antibody against peptide or KLH-peptide was determined by ELISA method.
Results: After repeated injection of CD166 peptide bound to KLH, antibody against CD166 peptide was produced, but it had a low titer. Most of the antibody produced was directed against the KLH-peptide binding site. Also, the tendency of produced antibodies to bind to KLH-peptide was higher compared to the peptide alone.
Conclusion: The produced antibodies were mostly against the antigenic index of KLH peptide. The synthetic and small size of the injected peptide can be the reason for the low titer and affinity of the antibody to it.
Type of Study: Research |
Subject:
ایمونولوژی
References
1. Ferragut F, Vachetta VS, Troncoso MF, Rabinovich GA, Elola MT. ALCAM/CD166: A pleiotropic mediator of cell adhesion, stemness and cancer progression. Cytokine Growth Factor Rev 2021;61:27-37. [DOI:10.1016/j.cytogfr.2021.07.001] [PMID]
2. Cardeñes B, Clares I, Bezos T, Toribio V, López-Martín S, Rocha A, et al. ALCAM/CD166 Is Involved in the Binding and Uptake of Cancer-Derived Extracellular Vesicles. Int J Mol Sci 2022;23(10). [DOI:10.3390/ijms23105753] [PMID] [PMCID]
3. Bowen MA, Bajorath J, D'Egidio M, Whitney GS, Palmer D, Kobarg J, et al. Characterization of mouse ALCAM (CD166): the CD6-binding domain is conserved in different homologs and mediates cross-species binding. Eur J Immunol 1997;27(6):1469-78. [DOI:10.1002/eji.1830270625] [PMID]
4. Cortés F, Deschaseaux F, Uchida N, Labastie MC, Friera AM, He D, et al. HCA, an immunoglobulin-like adhesion molecule present on the earliest human hematopoietic precursor cells, is also expressed by stromal cells in blood-forming tissues. Blood 1999a;93(3):826-37.
https://doi.org/10.1182/blood.V93.3.826 [DOI:10.1182/blood.v93.3.826] [PMID]
5. Brinkhof B, Zhang B, Cui Z, Ye H, Wang H. ALCAM (CD166) as a gene expression marker for human mesenchymal stromal cell characterisation. Gene X 2020;5:100031. [DOI:10.1016/j.gene.2020.100031] [PMCID]
6. Weichert W, Knösel T, Bellach J, Dietel M, Kristiansen G. ALCAM/CD166 is overexpressed in colorectal carcinoma and correlates with shortened patient survival. J Clin Path 2004;57(11):1160-64. [DOI:10.1136/jcp.2004.016238] [PMID] [PMCID]
7. Fujiwara K, Ohuchida K, Sada M, Horioka K, Ulrich III CD, Shindo K, et al. CD166/ALCAM expression is characteristic of tumorigenicity and invasive and migratory activities of pancreatic cancer cells. PLoS One 2014;9(9):e107247. [DOI:10.1371/journal.pone.0107247] [PMID] [PMCID]
8. Kim DK, Ham MH, Lee SY, Shin MJ, Kim YE, Song P, et al. CD166 promotes the cancer stem-like properties of primary epithelial ovarian cancer cells. BMB Rep 2020;53(12):622-27.
https://doi.org/10.5483/BMBRep.2020.53.12.102 [DOI:10.5483/bmbrep.2020.53.12.102] [PMID] [PMCID]
9. Kalantari E, Taheri T, Fata S, Abolhasani M, Mehrazma M, Madjd Z, et al. Significant co-expression of putative cancer stem cell markers, EpCAM and CD166, correlates with tumor stage and invasive behavior in colorectal cancer. World J Surg Oncol 2022;20(1):15. [DOI:10.1186/s12957-021-02469-y] [PMID] [PMCID]
10. Tachezy M, Zander H, Wolters-Eisfeld G, Müller J, Wicklein D, Gebauer F, et al. Activated leukocyte cell adhesion molecule (CD166): an "inert" cancer stem cell marker for non-small cell lung cancer? Stem Cells 2014;32(6):1429-36. [DOI:10.1002/stem.1665] [PMID]
11. Jezierska A, Matysiak W, Motyl T. ALCAM/CD166 protects breast cancer cells against apoptosis and autophagy. Med Sci Monit 2006;12(8):BR263-73. [Google Scholar]
12. Donizy P, Zietek M, Halon A, Leskiewicz M, Kozyra C, Matkowski R. Prognostic significance of ALCAM (CD166/MEMD) expression in cutaneous melanoma patients. Diagn Pathol 2015;10:86. [DOI:10.1186/s13000-015-0331-z] [PMID]
13. Ihnen M, Köhler N, Kersten J, Milde-Langosch K, Beck K, Höller S, et al. Expression levels of Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166) in primary breast carcinoma and distant breast cancer metastases. Dis Markers 2010;28(2):71-8. [DOI:10.1155/2010/812509] [PMID]
14. Swart GW. Activated leukocyte cell adhesion molecule (CD166/ALCAM): developmental and mechanistic aspects of cell clustering and cell migration. Eur J Cell Biol 2002;81(6):313-21. [DOI:10.1078/0171-9335-00256] [PMID]
15. Mezzanzanica D, Fabbi M, Bagnoli M, Staurengo S, Losa M, Balladore E, et al. Subcellular localization of activated leukocyte cell adhesion molecule is a molecular predictor of survival in ovarian carcinoma patients. Clin Cancer Res 2008;14(6):1726-33.
https://doi.org/10.1158/1078-0432.CCR-07-0428 [DOI:10.1158/1078-0432.ccr-07-0428] [PMID]
16. Erturk K, Tastekin D, Bilgin E, Serilmez M, Bozbey HU, Sakar B. Serum activated leukocyte cell adhesion molecule and intercellular adhesion molecule-1 in patients with gastric cancer: Can they be used as biomarkers? Biomed Pharmacother 2016;77:86-9. [DOI:10.1016/j.biopha.2015.12.006] [PMID]
17. Kulasingam V, Zheng Y, Soosaipillai A, Leon AE, Gion M, Diamandis EP. Activated leukocyte cell adhesion molecule: a novel biomarker for breast cancer. Int J Cancer 2009;125(1):9-14. [DOI:10.1002/ijc.24292] [PMID] [PMCID]
18. Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F. Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J 2005;46(3):258-68. [DOI:10.1093/ilar.46.3.258] [PMID]
19. Demlie T, Balcha E, Fesseha H. Monoclonal Antibody and its Diagnostic Application-Review. Biomed J Sci Techn Res 2020;30(4):23645-51. [DOI:10.26717/BJSTR.2020.30.004997]
20. Stapleton S, O' Kennedy R, Tully E. IMMUNOASSAYS | Production of Antibodies. In: Worsfold P, Townshend A, Poole C, editors. Encyclopedia of Analytical Science (Second Edition). Oxford: Elsevier; 2005. p. 306-16.
https://doi.org/10.1016/B0-12-369397-7/00263-6 [DOI:10.1016/b0-12-369397-7/00263-6]
21. Ascoli CA, Aggeler B. Overlooked benefits of using polyclonal antibodies. Biotechniques 2018;65(3):127-36. [DOI:10.2144/btn-2018-0065] [PMID]
22. Schunk MK, Macallum GE. Applications and optimization of immunization procedures. ILAR J 2005;46(3):241-57. [DOI:10.1093/ilar.46.3.241] [PMID]
23. Nakazawa M, Mukumoto M, Miyatake K. Production and Purification of Polyclonal Antibodies. High-Resolution Imaging of Cellular Proteins: Methods Protocols 2016:49-59. [DOI:10.1007/978-1-4939-6352-2_3] [PMID]
24. Stills HF. Polyclonal antibody production. The laboratory rabbit, Guinea pig, Hamster, and other rodents: Elsevier; 2012. p. 259-74.
https://doi.org/10.1016/B978-0-12-380920-9.00011-0 [DOI:10.1016/b978-0-12-380920-9.00011-0]
25. Bollen LS, Crowley A, Stodulski G, Hau J. Antibody production in rabbits and chickens immunized with human IgG A comparison of titre and avidity development in rabbit serum, chicken serum and egg yolk using three different adjuvants. J Immunol Methods 1996;191(2):113-20. [DOI:10.1016/0022-1759(96)00010-5] [PMID]
26. Hancock DC, O'Reilly NJ. Production of polyclonal antibodies in rabbits. Methods Mol Biol 2005;295:27-40. [DOI:10.1385/1-59259-873-0:027] [PMID]
27. Nakazawa M, Mukumoto M, Miyatake K. Production and Purification of Polyclonal Antibodies. Methods Mol Biol 2016;1474:49-59. [DOI:10.1007/978-1-4939-6352-2_3] [PMID]
28. Al-Tubuly AA. SDS-PAGE and Western Blotting. Methods Mol Med 2000;40:391-405. [DOI:10.1385/1-59259-076-4:391] [PMID]
29. Geerligs HJ, Weijer WJ, Bloemhoff W, Welling GW, Welling-Wester S. The influence of pH and ionic strength on the coating of peptides of herpes simplex virus type 1 in an enzyme-linked immunosorbent assay. J Immunol Methods 1988;106(2):239-44. [DOI:10.1016/0022-1759(88)90203-7] [PMID]
30. Doucet J, Zhao A, Fu J, Avrameas A. Development and validation of an ELISA at acidic pH for the quantitative determination of IL-13 in human plasma and serum. Dis Markers 2013;35(5):465-74. [DOI:10.1155/2013/290670] [PMID] [PMCID]
31. Li Q, Gordon M, Cao C, Ugen KE, Morgan D. Improvement of a low pH antigen-antibody dissociation procedure for ELISA measurement of circulating anti-Abeta antibodies. BMC Neurosci 2007;8:22. [DOI:10.1186/1471-2202-8-22] [PMID] [PMCID]
32. 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]
33. Camacho CJ, Katsumata Y, Ascherman DP. Structural and thermodynamic approach to peptide immunogenicity. PLoS Comput Biol 2008;4(11):e1000231. [DOI:10.1371/journal.pcbi.1000231] [PMID] [PMCID]
34. Amini N, Vishteh MN, Zarei O, Hadavi R, Ahmadvand N, Rabbani H, et al. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein. Iran J Basic Med Sci 2014;17(6):396-400. [DOI:10.1016/j.clinbiochem.2011.08.018]
35. Razavi H, Teimoori A, Saberfar E, Soleimanjahi H, Goodarzi Z. Peptide based polyclonal antibody production against bovine rotavirus non structure protein4 (NSP4). Archives of Razi Institute 2015;70(3):157-61. [Google Scholar]
Send email to the article author
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
