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International Journal of Medical Laboratory
Shahid Sadoughi University of Medical Sciences
Thu, Apr 25, 2024
[Archive]
Volume 10, Issue 1 (February 2023)
IJML 2023, 10(1): 67-74 |
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Zaker E, Zare F, Hejazi S H, Khanahmad H, Kalantar S M. A Mouse Monoclonal Antibody Against Human IFN-γ and its Characters. IJML 2023; 10 (1) :67-74
URL: http://ijml.ssu.ac.ir/article-1-464-en.html
URL: http://ijml.ssu.ac.ir/article-1-464-en.html
Department of Medical Genetics, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Department of Genetics and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Abstract: (188 Views)
Background and Aims: A monoclonal antibody (mAb) can unambiguously identify, quantify, and purify an antigen or particular epitope at a large scale. The superiority of these antibodies lies in their specificity for the antigenic determinant. So, this study aims to prepare mouse mAb-secreting hybridoma against human gamma interferon (IFN-γ) and determine the produced antibody's characters.
Materials and Methods: Mouse splenic B lymphocytes immunized with recombinant human IFN-γ were fused with mouse SP2/0 cells. The hybridized cells were selected by hypoxanthine-aminopterin-thymidine and hypoxanthine-thymidine media to obtain monoclonal antibody-producing hybridoma cells. Finally, indirect enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and western blot were used to confirm the creation of antibody-secreting hybridoma cells.
Results: mAb against IFN-γ were produced by fusing SP2/0 mouse non-secretory myeloma cell line with the spleen cells of immunized mice. This antibody's indirect ELISA optical density was 2.055 on average, and the desired antibody bands were confirmed in SDS-PAGE compared to Septicol® (commercial antibody). Also, in the western blot, the desired antibody could bind to the antigen. IFN-γ transferred on nitrocellulose membrane. In ELISA and western blot tests, anti-mouse IgG conjugated antibodies were used; therefore, the mAb IgG isotype was taken into consideration.
Conclusion: In this study, a mouse mAb was obtained by immunization of Balb/C mice and fusion of spleen cells of these mice with the SP2/0 cells, which can specifically bind to recombinant human IFN-γ and can be used to detect IFN-γ secretion in all types of intracellular infections, including latent tuberculosis.
Materials and Methods: Mouse splenic B lymphocytes immunized with recombinant human IFN-γ were fused with mouse SP2/0 cells. The hybridized cells were selected by hypoxanthine-aminopterin-thymidine and hypoxanthine-thymidine media to obtain monoclonal antibody-producing hybridoma cells. Finally, indirect enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and western blot were used to confirm the creation of antibody-secreting hybridoma cells.
Results: mAb against IFN-γ were produced by fusing SP2/0 mouse non-secretory myeloma cell line with the spleen cells of immunized mice. This antibody's indirect ELISA optical density was 2.055 on average, and the desired antibody bands were confirmed in SDS-PAGE compared to Septicol® (commercial antibody). Also, in the western blot, the desired antibody could bind to the antigen. IFN-γ transferred on nitrocellulose membrane. In ELISA and western blot tests, anti-mouse IgG conjugated antibodies were used; therefore, the mAb IgG isotype was taken into consideration.
Conclusion: In this study, a mouse mAb was obtained by immunization of Balb/C mice and fusion of spleen cells of these mice with the SP2/0 cells, which can specifically bind to recombinant human IFN-γ and can be used to detect IFN-γ secretion in all types of intracellular infections, including latent tuberculosis.
Type of Study: Research |
Subject:
Immunology
Received: 2022/12/1 | Accepted: 2023/01/15 | Published: 2023/03/1
Received: 2022/12/1 | Accepted: 2023/01/15 | Published: 2023/03/1
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