A Mouse Monoclonal Antibody Against Human IFN-γ  and its Characters

Erfan  Zaker; Fateme  Zare; Seyed Hossein  Hejazi; Hossein  Khanahmad; Seyed Mehdi  Kalantar

doi:10.18502/ijml.v10i1.12427

Erfan Zaker Department of Medical Genetics, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Fateme Zare Reproductive Immunology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Seyed Hossein Hejazi Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Hossein Khanahmad Department of Genetics and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Seyed Mehdi Kalantar Department of Medical Genetics, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

DOI: https://doi.org/10.18502/ijml.v10i1.12427

Keywords: Hybridoma, IFN-γ, Monoclonal antibody, SP2/0

Abstract

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.