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کاربرد نوع شرط:
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: Apoptosis,Chronic Lymphocytic Leukemia,Monoclonal antibody,Fibromodulin (FMOD),Glycosylphosphatidylinositol
- چکیده:
- چکیده انگلیسی: Background: We have previously reported the aberrant expression of Fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). Although FMOD has been considered as a cytoplasmic or secretory protein, we discovered the cell surface expression of FMOD in leukemic B cells via anchoring with glycosylphosphatidylinositol (GPI). Objective: To evaluate FMOD as a new biomarker in CLL patients in comparison with healthy individuals. Methods: A monoclonal antibody was generated against human FMOD. The cell surface expression of FMOD in 52 CLL patients and 45 healthy individuals were compared by flow cytometry. A bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) was used to determine the cell surface localization of FMOD using ELISA and flow cytometry techniques. Annexin V-FITC and propidium iodide (PI) was used to detect apoptosis induction in CLL PBMCs following in vitro incubation with anti-FMOD mAb. Results: The results demonstrated the widespread cell surface expression of GPI-anchored FMOD in CLL patients (median: 79.9 %), although healthy individuals had low FMOD expression (median: 6.2 %) (p≤0.0001). The cut-off value of FMOD expression was estimated with high sensitivity and specificity at 17.9%. Furthermore, in vitro apoptosis induction of leukemic cells following incubation with anti-FMOD mAb showed a direct apoptosis of CLL cells (27.9%) with very low effect on healthy PBMCs (6%). Conclusion: The membrane-anchoring of FMOD by means of a GPI moiety in leukemic cells supports FMOD as a highly potential diagnostic and therapeutic target in CLL patients.
- انتشار مقاله: 15-07-1397
- نویسندگان: Lia Farahi,Fatemeh Ghaemimanesh,Saeideh Milani,Seyed Mohsen Razavi,Reza Hadavi,Ali Ahmad Bayat,Ali Salimi,Mohammad Mehdi Akhondi,Hodjattallah Rabbani
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: ELISA,Flow cytometry,CD34,Monoclonal antibody,Western blotting
- چکیده:
- چکیده انگلیسی: 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.- انتشار مقاله: 15-05-1395
- نویسندگان: Mahmood Shams,Mahmood Jeddi-Tehrani,Farzaneh Notash Haghighat,Ali Ahmad Bayat,Jafar Mahmoudian,Mohammad Reza Rezvani
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Biotechnology
- نوع مقاله: Journal Article
- کلمات کلیدی: Leukemia, Lymphocytic, Chronic, B-Cell,Fibromodulin,Antibodies, Monoclonal
- چکیده:
- چکیده انگلیسی: Background: The unique expression of fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL) has been previously reported. Detecting FMOD in CLL patients using specific anti-FMOD mAbs might provide a promising method in detection, monitoring, and prognosis of CLL.
Objectives: In this study, we aimed for producing specific antibodies against FMOD to facilitate further cohort study of CLL, thus addressing FMOD as a potential target of detection.
Materials and Methods: Human FMOD gene (1087 bp) was extracted from genome of the CLL patients, and was cloned into the expression vector of pET-22b (+). The recombinant FMOD protein (rFMOD) was expressed in Escherichia coli. The purified rFMOD protein was used as an immunogen in rabbit and mice. Hybridoma technology was used to develop the monoclonal antibodies (mAbs). Polyclonal antibody (pAb) was purified from the rabbit sera using affinity column. The reactivity of anti-FMOD antibodies was assessed in ELISA, immunocytochemistry (ICC) and Western blot.
Results: ICC results showed that the anti-FMOD antibodies specifically detected FMOD in CLL PBMCs and cell lines. The developed anti-FMOD pAb detected FMOD in CLL lysates, compared to healthy PBMCs, in Western blot and ELISA.
Conclusions: The developed anti-FMOD mAbs, and pAb specifically detect FMOD in CLL samples and might be used as research tools for further investigations in CLL.- انتشار مقاله: 09-03-1397
- نویسندگان: Lia Farahi,Fatemeh Ghaemimanesh,Saeideh Milani,Seyed Mohsen Razavi,Ali Ahmad Bayat,Hodjattallah Rabbani,Mohammad Mehdi Akhondi
- مشاهده