Production and characterization of murine monoclonal antibodies against human synthetic peptide of CD34
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1
Immunology Research Center, Iran
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2
Department of Immunology, Faculty of Medicine, Tabriz University of Medical Science, Tabriz, Iran, Iran
The treatment of hematologic malignancies, solid tumors, and some non-malignant diseases like immunodeficiency diseases is offered by hematopoietic stem cells (HSCs) as a unique self-renewal and differentiation source which most commonly is selected by CD34 surface marker for HSC. By the advent of hybridoma technology, monoclonal antibodies applications exert extensive changes in medical fields such as diagnosis, treatment and purification. Antibodies are highly specific and functional in applications such as ELISA, Flowcytometry, Immunocytochemistry, and Western blot assays. In this study, splenocytes from Balb/c mice immunized with two 14-mer synthetic peptide were fused with SP2/0 murine myeloma cells. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium. Supernatant of hybridoma cells were screened by enzyme-linked immunosorbent assay (ELISA) for antibody production and the desired clones were selected and cloned by limiting dilution (L.D) assay. Eventually, the specificity and cross reactivity of secreted mAbs were further studied by ELISA and western blot analysis (WB). Isotype of this mAb was identified as γ heavy chain and k light chain. Reactivity of these antibodies was then evaluated in different immunological assays including ELISA, Immunofluorescence (IF), Western blot (WB) and Flowcytometry. Monoclonal antibody was purified by affinity chromatography on protein A-Sepharose and then confirmed by SDS-PAGE. These results indicate that peptide-based antibody against CD34 surface marker of HSC can be used for research, diagnosis and purification.
Keywords:
Monoclonal antibody,
synthetic peptide,
CD34,
murine,
ELISA
Conference:
15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.
Presentation Type:
Abstract
Topic:
Translational immunology and immune intervention
Citation:
Aghebati Maleki
L,
Majidi
J,
Baradaran
B,
Abdolalizadeh
J and
Movassaghpour
A
(2013). Production and characterization of murine monoclonal antibodies against human synthetic peptide of CD34.
Front. Immunol.
Conference Abstract:
15th International Congress of Immunology (ICI).
doi: 10.3389/conf.fimmu.2013.02.00485
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Received:
31 Mar 2013;
Published Online:
22 Aug 2013.
*
Correspondence:
Dr. Behzad Baradaran, Immunology Research Center, Tabriz, Iran, Behzad_im@yahoo.com