Fatemeh Hajighasemi
Volume 8, Issue 2 , June 2011, , Pages 120-126
Abstract
Background: Leukemia is a malignant proliferative disorder of the hematopoietic cells. The important role of angiogenesis in leukemia has been reported by several studies. Matrix metalloproteinases (MMPs) are a large group of endopeptidases which degredate the extracellular matrix and play an important ...
Read More
Background: Leukemia is a malignant proliferative disorder of the hematopoietic cells. The important role of angiogenesis in leukemia has been reported by several studies. Matrix metalloproteinases (MMPs) are a large group of endopeptidases which degredate the extracellular matrix and play an important role in angiogenesis. Objective: The present study was conducted to evaluate the patterns of MMP-2 activity in three leukemic cell lines. Methods: Human leukemic monocyte (U937) and T cells (Molt-4 and Jurkat) were cultured in complete RPMI-1640 medium. The cells were then seeded at a density of 106 cells/ml and were incubated with different concentrations of phorbol myristate acetate (PMA) (1-25 ng/ml) or phytoheamagglutinin (PHA) (2-10 μg/ml) for 24 hours. The MMP-2 activity in cell-conditioned media was then evaluated by gelatin zymography. Statistical comparisons between groups were made by analysis of variance (ANOVA). Results: PHA/PMA significantly and dose-dependently increased MMP-2 activity in U937 cells after 24 hours of incubation compared with untreated control cells. Moreover, PHA/PMA significantly induced MMP-2 activity in Molt-4 and Jurkat cells after 24 hours of incubation in a dose-dependent manner compared with untreated control cells. Conclusion: We conclude that human leukemic Jurkat, U937 and Molt-4 cells could potentially display MMP-2 activity with different degrees. Thus, these cell lines could provide an appropriate system to study the mechanisms regulating MMPs production in leukemia patients.
Fatemeh Hajighasemi; Soheila Gharagozlou; Nasrin Moheghi; Roya Ghods; Jalal Khoshnoodi; Fazel Shokri
Volume 2, Issue 3 , September 2005, , Pages 125-133
Abstract
Background: There are two subclasses of human IgA (IgA1 and IgA2) that differ in antigenic properties and in chemical composition. The constant domains of α1 and α2 heavy chains have >95% sequence homology though major structural differences exist in the hinge region. Quantitation of IgA ...
Read More
Background: There are two subclasses of human IgA (IgA1 and IgA2) that differ in antigenic properties and in chemical composition. The constant domains of α1 and α2 heavy chains have >95% sequence homology though major structural differences exist in the hinge region. Quantitation of IgA subclass levels depends on the availability of monoclonal antibodies (MAbs) specific for conserved conformational or linear epitopes restricted to each subclass. Objective: To produce, select and characterize monoclonal antibodies specific for human IgA2. Methods: Splenocytes from BALB/C mice immunized with a human IgA2 myeloma protein were fused with SP2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody (Ab) secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins and some animal sera by ELISA and immunoblotting. The affinity constant (Kaff) was also determined by ELISA. Results: Four murine hybridoma clones designated 2F20G5, 2F20B5, 3F20E3 and 6F20H11 were obtained that secreted MAbs specific for the human IgA2. 2F20G5 and 6F20H11 MAbs react with linear epitope(s) while 2F20B5 and 3F20E3 react with conformational epitope(s) located to human IgA2 subclass. 2F20G5 MAb displays a weak cross-reactivity with monkey and rabbit sera and a strong cross-reactivity with cat and dog sera while the other three MAbs showed no cross-reactivity with the animal sera tested. Conclusion: These MAbs, especially 6F20H11 with high affinity constant (6.03 ×109 M-1) are useful tools for quantitation of human IgA2 subclass levels in various diseases. Cross-reactivity of 2F20G5 MAb with some animalsera suggests phylogenic conservation ofthe epitope recognized by this MAb.
Fatemeh Hajighasemi; Ali Akbar Saboor-Yaraghi; Fazel Shokri
Volume 1, Issue 3 , December 2004, , Pages 154-161
Abstract
Background: The affinity of an antibody to its antigen is a crucial parameter in its biological activity and performance of an immunoassay such as ELISA. Affinity of most IgG specific MAbs are often determined by methods which require labeling of either antigen or antibody, and are sometimes difficult ...
Read More
Background: The affinity of an antibody to its antigen is a crucial parameter in its biological activity and performance of an immunoassay such as ELISA. Affinity of most IgG specific MAbs are often determined by methods which require labeling of either antigen or antibody, and are sometimes difficult to control, do not always lead to the expected signal and often result in immunological modification of the molecules. Moreover, direct solid phase binding assays pose some problems such as diffusion effects and difficulties in reaching equilibrium due to heterogeneous binding and co-operativity. Objective: To employ a rapid and simple ELISA-based method for measuring affinity constants of two pan-h-IgG specific MAbs (3F2D8 and 5F19G11) established in our laboratory. Methods: The method is based on the effect of antibody affinity on the sigmoidal dose response curve. In this method, the binding of anti-human IgG (anti-h-IgG) MAbs with their corresponding antigen was measured using serial concentrations of both antigen and antibody. The amount of antibody bound to the antigen on the plate is represented as a sigmoidal curve of OD versus the logarithm of antibody concentration added to each well. Results: Based on the data obtained from this study, the affinity constants of 3F2D8 and 5F19G11 MAbs were 0.74 x 10 8 Mol –1 and 0.96 x 10 7 Mol –1, respectively. Conclusion: 3F2D8 MAb with reasonably high affinity is suggested as a candidate for quantitative measurement of IgG by ELISA, whereas 5F19G11 MAb could be considered as a suitable tool for immunoaffinity chromatography.
