Document Type : Applied Immunology Series
Authors
- Mahmood Shams 1, 2
- Mahmood Jeddi-Tehrani 3
- Farzaneh Notash Haghighat 3
- Ali Ahmad Bayat 3
- Jafar Mahmoudian 3
- Mohammad Reza Rezvani 1, 2, , 4
1 Department of Hematology, Faculty of Allied Medicine, Tehran University of Medical Sciences
2 Department of Hematology, Faculty of Allied Medicine, Iran University of Medical Sciences
3 Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
4 Immune and Gene Therapy Laboratory, Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden
Abstract
Background: Human CD34 is a transmembrane glycoprotein which is expressed in human hematopoietic stem cells (HSCs) and the small- vessel endothelial cells of a variety of tissues. CD34 plays a critical role as a marker for diagnosis and classification of leukemia. Anti CD34 antibodies are used for isolation and purification of HSCs from bone marrow, peripheral blood and cord blood.
Objective: To characterize a newly produced monoclonal antibody against a human CD34 peptide.
Methods: Anti CD34 monoclonal antibody (Clone 2C10-D3) was purified from mouse ascitic fluid and hybridoma cell culture supernatants by affinity chromatography and its immune reactivity was examined by ELISA. The purified antibody was further characterized using Western blot and flow cytometry on TF1 (Human Erythroblast) cell line.
Results: ELISA experiment revealed that the antibody recognized CD34 peptide. Western blot analysis on TF1 cell lysate confirmed the reactivity of the antibody with a 42 KDa protein. Blocking the antibody with a saturating concentration of specific CD34 peptide resulted in loss of its activity with TF1 lysate in Western blot. The 2C10-D3 antibody reacted with TF1 cells in flow cytometry in a similar manner to a commercial anti CD34 monoclonal antibody.
Conclusions: Our data suggest that the anti CD34 monoclonal antibody (Clone 2C10-D3) is an appropriate antibody to study the CD34+ cells by flow cytometry and Western blot.
Keywords
)