A methodological approach for production and purification of polyclonal ‎antibody against dog IgG ‎

Document Type : Original Article

Authors

1 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

2 Department of Immunology, Faculty of Medicine, Tabriz ‎University of Medical Sciences, Tabriz, Iran

3 Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

Abstract

Antibodies are a class of biomolecules that has an important role in the immune system and lots of applications in biotechnological methods and in pharmaceutics. Production and purification of antibodies in laboratory animals is one of the first ways to manufacture of these prominent tools. The obtained antibodies from these process could be used in various types of bioassay techniques such as enzyme linked immunosorbent assay (ELISA), radioimmunoassay, etc. Also, antibodies employed in diagnostics applications in humans and other animals in order to detect specific antigens.In this study, we aimed to produce and purify anti-dog IgG via immunizing rabbits with dog IgG in combination with Freund's adjuvant. Polyclonal IgG were purified by ion exchange chromatography and then the purified antibody was labeled with horse radish peroxidase (HPR). Direct ELISA was used to determine the optimum titer and cross-reactivity of HRP conjugated IgG. The purity of various IgG preparations and the optimum dilution of prepared HRP conjugated IgG, respectively, was about 95.00% and 1:8000. This study showed that efficiency ion-exchange chromatography could be an appropriate method for purification of IgG antibodies. This antibody could be a useful tool for future dog immune diagnosis tests. This product characterization shown here sets the foundations for future work on dog IgGs.

Keywords

Main Subjects


  1.  

    1. Eivazi S, Majidi, Aghebati-Maleki L, et al. Production and purification of a polyclonal antibody against purified mouse IgG2b in rabbits towards designing mouse monoclonal isotyping kits. Adv Pharm Bull 2015; 5(1): 109-113.
    2. Huse K, Bohme HJ, Scholz GH. Purification of antibodies by affinity chromatography. J Biochem Biophys Methods. 2002;51(3): 217-231.
    3. Maleki LA, Majidi J, Baradaran B, et al. Production and characterization of murine monoclonal antibody against synthetic peptide of CD34. Hum Antibodies 2013; 22(1-2): 1-8.
    4. Robinson MK, Weiner LM, Adams GP. Improving monoclonal antibodies for cancer therapy. Drug Dev Res 2004; 61(3): 172-187.
    5. Heddle RJ, Rowley D. Dog immunoglobulins. I. immunochemical characterization of dog serum, parotid saliva, colostrum, milk and small bowel fluid. Immunology. 1975; 29(1):185-195.
    6. Aghebati-Maleki L, Majidi J, Baradaran B, et al.  Generation and characterization of anti-CD34 monoclonal antibodies that react with hematopoietic stem cells. Cell J 2014; 16(3): 361-366.
    7. Tishchenko GA, Bleha M, Skvor J, et al. Effect of salt concentration gradient on separation of different types of specific immunoglobulins by ion-exchange chromatography on DEAE cellulose. J Chromatogr B Biomed Sci Appl 1998; 706(1): 157-166.
    8. Verdoliva A, Basile G, Fassina G. Affinity purification of immunoglobulinss from chicken egg yolk using a new synthetic ligand. J Chromatogr B Biomed Sci Appl 2000; 749(2):233-242.
    9. Aghebati-Maleki L, Majidi J, Baradaran B, et al. Large scale generation and characterization of anti-human CD34 monoclonal antibody in ascetic fluid of Balb/c Mice. Adv Pharm Bull 2013;3(1):211-216.
    10. Waner T, Harrus S, Jongejan F, et al. Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia canis. Vet Parasitol 2001; 95(1):1-15.
    11. Ezzatifar f, Majidi j, Baradaran B, et al. Large scale generation and characterization of anti-Human IgA monoclonal antibody in ascitic fluid of Balb/c mice. Adv Pharm Bull. 2015; 5(1): 97-102.
    12. Sepehr KS, Baradaran B, Majidi J, et al. Development and characterization of monoclonal antibodies against human CD20 in Balb/c mice. Hum Antibodies 2012; 21(3-4):57-64.
    13. Raoufinia R, Mota A, Nozari S, et al. A methodological approach for purification and characterization of human serum albumin. J Immunoassay Immunochem 2016; 37(6):623-635.
    14. Maleki LA, Baradaran B, Majidi J, et al. Future prospects of monoclonal antibodies as magic bullets in immuno-therapy. Hum Antibodies 2013; 22(1-2):9-13.
    15. Blakesley RW, Boezi JA. A new staining technique for proteins in polyacrylamide gels using Coomassi Brilliant Blue G-250. Anal Biochem 1977; 82: 580-582.
    16. Moradi Nebrin Z, Majidi J, Aghebati-Maleki L, et al. Polyclonal antibody production against mouse purified IgG2a towards use in basic research. Res Mol Med 2016, 4(1): 30-35.                             
    17. Arce C, Moreno A, Millán Y, et al. Production and characterization of monoclonal antibodies against dog immunoglobulin isotypes. Vet Immunol Immunopathol 2002 ;88(1-2): 31-41.
    18. Di Febo T, Luciani M, Ciarelli A, et al. Production and characterization of monoclonal antibodies against horse immunoglobulins useful for the diagnosis of equine diseases. J Immunoassay Immunochem 2015; 36(3): 253-264.
    19. Aghebati-Maleki L, Bakhshinejad B, Baradaran B, et al. Phage display as a promising approach for vaccine development. J Biomed Sci 2016; 23(1): 66.
    20. Aghebati-Maleki L, Baradaran B, Abdolalizadeh J, et al. A unique report: Development of super anti-human IgG monoclone with optical density over than 3. Adv Pharm Bull 2013; 3(2): 333-337.
    21. Aghebati-Maleki L, Younesi V, Jadidi-Niaragh F, et al. Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique. Hum Antibodies 2017; 25(1-2):57-63.
    22. Aghebati-Maleki L, Younesi V, Baradaran B, et al. Antiproliferative and apoptotic effects of novel anti-ROR1 single-chain antibodies in hematological malignancies. SLAS Discov 2017; 22(4): 408-417.
    23. Ezzatifar F, Majidi J, Baradaran B, et al. Development and characterization of monoclonal antibodies against human IgA in Balb/c mice. Hum Antibodies 2014; 23(1-2): 7-12.
    24. Mejías M, Molina MC, Ferreira A. Assessment of the IgG index in dogs by indirect immunoenzimatic assays as diagnostic tool for inflammatory diseases of central nervous system. Res Vet Sci 2008;84(3): 335-340.
    25. Bergeron LM, McCandless EE, Dunham S, et al. Comparative functional characterization of canine IgG subclasses. Vet Immunol Immunopathol 2014 ;157(1-2):31-41.