Jalal Abdolalizadeh
Tabriz University Of Medical Sciences, Lab Sciences, Faculty Member
- Proteomics, Two-Dimensional Gel Electrophoresis, Western blotting, Phage display, Monoclonal Antibody, Protein Purification, and 7 moreImmunology, Monoclonal Antibodies, Conjugation, Biochemistry, Chromatography, Protein Science, and Protein Engineering(Immunology, Monoclonal Antibodies, Conjugation, Biochemistry, Chromatography, Protein Science, and Protein Engineering)edit
- Tabriz University of Medical Sciences-Tabriz-Iranedit
There is increasing evidence that vascular endothelial growth factor (VEGF) has a role in the development of vascular leakage in the hypoxic brain. Our recent work showed an anti-edematous effect of ghrelin on brain hypoxia. However, the... more
There is increasing evidence that vascular endothelial growth factor (VEGF) has a role in the development of vascular leakage in the hypoxic brain. Our recent work showed an anti-edematous effect of ghrelin on brain hypoxia. However, the underlying mechanisms by which ghrelin exerts its anti-edematous effect are still unclear. For this purpose, we examined the effects of ghrelin on VEGF expression in the hypoxic brain. Adult male Wistar rats were divided into acute or chronic controls, acute or chronic hypoxia, and ghrelin-treated acute or chronic hypoxia groups. Systemic hypoxia was induced in rats by a hypoxic chamber (O2 10-11 %) for 2 days (acute) or 10 days (chronic). Effects of ghrelin on VEGF expression were evaluated using immunoblotting. Our data revealed that acute and chronic hypoxia increased VEGF expression in both acute and chronic hypoxia (P < 0.001). Ghrelin significantly reduced this expression in hypoxic conditions (P < 0.001). Our findings demonstrated the b...
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CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have... more
CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs).
Research Interests: Cell()
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Research Interests: Medical()
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It is estimated that one third of the world's population were infected with M. tuberculosis, but only 10% of them have developed in to disease form. This subject refers to differences in host immune system activity against the... more
It is estimated that one third of the world's population were infected with M. tuberculosis, but only 10% of them have developed in to disease form. This subject refers to differences in host immune system activity against the tuberculosis. Vitamin D and its receptor (VDR) are important factors in the host innate immune system against the tuberculosis. In the present study VDR gene polymorphisms and its relationship with plasma vitamin D levels in susceptibility to tuberculosis have been investigated. The subjects were 84 patients with tuberculosis and 90 healthy controls. Vitamin D levels were measured in all study participants. DNA was isolated from the blood leukocytes of all groups and amplified by polymerase chain reaction (PCR). Then restriction fragment length polymorphism (RFLP) was performed on each PCR products to study the VDR gene polymorphisms. The statistical analyses were conducted using SPSS. There was no statistically significant relationship between polymorphis...
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CD14 is a myeloid differentiation antigen expressed primarily on peripheral blood monocytes, dendritic cells and macrophages. It is a key regulator of inflammatory responses to gram-negative bacteria, oxidative burst and septic shock. The... more
CD14 is a myeloid differentiation antigen expressed primarily on peripheral blood monocytes, dendritic cells and macrophages. It is a key regulator of inflammatory responses to gram-negative bacteria, oxidative burst and septic shock. The aim of this study was to produce and characterize monoclonal antibody against CD14 for use in detection and diagnosis of monocytes. To produce MAb against CD14 protein, mice were immunized with two KLH-conjugated CD14 peptides. The spleen cells of the immunized mice were then fused with SP2/0 by hybridoma technique. Fused cells were grown in selective medium and cloned by limiting dilution method. The desired clones were selected and supernatants of hybridoma cells were screened by ELISA for antibody. Monoclonal antibody was purified by chromatography and confirmed by SDS-PAGE. Finally, immunoblotting and flowcytometry were recruited to explore the specificity of the MAb. Our results showed successful production and characterization of anti CD14 mo...
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CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have... more
CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with...
Research Interests: Cell()
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The treatment of hematologic malignancies and immunodeficiency diseases are 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. The... more
The treatment of hematologic malignancies and immunodeficiency diseases are 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. The purpose of this study was to develop and characterize monoclonal antibody against CD34 antigen for detection of hematopoietic stem cells. Balb/c mice were immunized with two synthetic peptides of CD34 and Spleen cells were fused with SP2/0.Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by limiting dilution. Large scale of monoclonal antibodies was produced by mouse ascites production of mAb (in vivo) method. Monoclonal antibody was purified by chromatography. Then reactivity of these antibodies was evaluated in different immunological assays including ELISA, immunofluorescence (IF), western blot (WB) and flowcytometry. In this study, between five positive clone wells, two clones were chosen for ...
Helicobacter pylori (HP) is a spiral-shaped microaerophilic gramnegative bacterium causes acute and chronic gastritis, duodenal ulcer and gastric cancer in human. Isolation and identification of specific antigenic proteins is of great... more
Helicobacter pylori (HP) is a spiral-shaped microaerophilic gramnegative bacterium causes acute and chronic gastritis, duodenal ulcer and gastric cancer in human. Isolation and identification of specific antigenic proteins is of great significance in better understanding of HP pathogenecity and selection of suitable antigen candidate for diagnosis and vaccine purposes. In this study protein pattern of 40 HP samples isolated from patients suffering from gastritis, cancer, duodenal ulcer and infected individuals without clinical symptoms was evaluated by Isoelectric focusing (IEF), Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE). Protein bands were stained with silver nitrate or Coomassie blue. Results of this study showed that protein patterns of members of each patient group (except gastritis patients) are similar significantly and have obvious differences among different groups. Apparently HP strains isolated from ...
Research Interests: Medical()
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Hypoxia Inducible Factor-1 plays a key transcriptional role in the adaptation of hypoxic solid tumors to low oxygen environment. Here, we aimed to investigate galbanic acid (GBA) inhibitory effects on HIF-1 activation during hypoxia and... more
Hypoxia Inducible Factor-1 plays a key transcriptional role in the adaptation of hypoxic solid tumors to low oxygen environment. Here, we aimed to investigate galbanic acid (GBA) inhibitory effects on HIF-1 activation during hypoxia and normoxia. MTT survival and Annexin V assays were used to evaluate GBA cytotoxicity and apoptosis in treated cells. Quantitative real time PCR and western blotting were used to estimate mRNA expression and translated protein, respectively. Results showed that GBA dose- and time-dependently decreased the in vitro growth of OVCAR-3 human epithelial carcinoma cells with an IC50 of approximately 37, 12.1 and 10μM GBA at 24, 48 and 72h, respectively. Following phosphatidylserine of outer leaflet of the plasma membrane revealed occurrence of early/late apoptosis in GBA treated cells. In addition, we found that GBA down-regulates HIF-1α and HIF-1β mRNA expression in both hypoxia and normoxia. To determine the mechanism of action, we showed that GBA did not inhibit Akt and EGFR mRNA expression, yet protein degradation investigation showed that GBA shortened the half-life of EGFR through decreasing its stability with a decrease of nearly 2 and 3h in A549 and OVCAR-3 cell lines, respectively. We also found that downstream genes contributed in glycolysis, including Eno 1 and GluT-1, are underexpressed in GBA treated cells in hypoxia. Conclusively, GBA may inhibit HIF-1 activation through down-regulation of its subunit expression in hypoxia, and increasing of EGFR degradation in normoxia.
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Monoclonal antibodies are key reagents that are used in biomedical researches, diagnosis of immunodeficiency diseases such as IgG subclasses deficiency and treatment of diseases like infections and cancers .For large scale production of... more
Monoclonal antibodies are key reagents that are used in biomedical researches, diagnosis of immunodeficiency diseases such as IgG subclasses deficiency and treatment of diseases like infections and cancers .For large scale production of monoclonal antibody, hybridoma ...
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To cite this article: Peyman Eivazy, Fatemeh Atyabi, Farhad Jadidi-Niaragh, Leili Aghebati Maleki, Abolfazl Miahipour, Jalal Abdolalizadeh & Mehdi Yousefi (2016): The impact of the codelivery of drug-siRNA by trimethyl chitosan... more
To cite this article: Peyman Eivazy, Fatemeh Atyabi, Farhad Jadidi-Niaragh, Leili Aghebati Maleki, Abolfazl Miahipour, Jalal Abdolalizadeh & Mehdi Yousefi (2016): The impact of the codelivery of drug-siRNA by trimethyl chitosan nanoparticles on the efficacy of chemotherapy for metastatic breast cancer cell line (MDA-MB-231), Artificial Cells, Nanomedicine, and Biotechnology,
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Background: The ability of polyclonal antibodies to react with many epitopes of an antigen makes them valuable reagents in research and diagnosis. The aim of this study was purification of mouse IgG2a and production of polyclonal antibody... more
Background: The ability of polyclonal antibodies to react with many epitopes of an antigen makes them valuable reagents in research and diagnosis. The aim of this study was purification of mouse IgG2a and production of polyclonal antibody against purified mouse IgG2a subclass. Materials and Methods: Mouse IgG2a was purified by ProA affinity. Verification method of the purified antibody was SDS-PAGE and ELISA by a mouse isotyping Kit. Rabbit was immunized with purified IgG2a. The production of antibody in rabbit was investigated by direct ELISA method. Rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. Polyclonal antibody was purified by ion-exchange chromatography and labeled with HRP. The titre and cross reactivity of product was detected by direct ELISA method. Results: The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 50-KDa, 25-30 KDa MW and a distinct band with 150 KDa MW. Isotype determination showed the presence of mouse IgG2a in related fraction. The titer of Anti-mouse polyclonal antibody was 200000. The optimum titer of prepared HRP conjugated IgG was 4000. Conjugated rabbit IgG has more cross reactivity with mouse IgG2b. Conclusion: Taking together, affinity chromatography and ion-exchange chromatography are appropriate techniques for purification of mouse IgG subclasses and rabbit IgG, respectively.
Research Interests: Antibody()
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Background: Recombinant human endostatin (rhES) is an angiogenesis inhibitor used as a specific drug for the treatment of non-small-cell lung cancer. As mRNA concentration affects the recombinant protein expression level, any factor... more
Background: Recombinant human endostatin (rhES) is an angiogenesis inhibitor used as a specific drug for the treatment of non-small-cell lung cancer. As mRNA concentration affects the recombinant protein expression level, any factor affecting mRNA concentration can alter the protein expression level. Response surface methodology (RSM) based on the Box-Behnken design (BBD) is a statistical tool for experimental design and for optimizing biotechnological processes.
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Glomalin (a glycoprotein) is produced by arbuscular mycorrhizal fungi and has the ability to sequester toxic heavy metals. We hypothesized that an increase in the concentration of Pb would lead to increased glomalin production and... more
Glomalin (a glycoprotein) is produced by arbuscular mycorrhizal fungi and has the ability to sequester toxic heavy metals. We hypothesized that an increase in the concentration of Pb would lead to increased glomalin production and consequently higher Pb-sequestration. In a two compartment pot culture experiment, clover plants (Trifolium repens L.) were inoculated with arbuscular mycorrhizal fungus, Rhizophagus irregularis, in sterile washed sand and then treated with Pb concentrations of 0, 150, 300 and 450 mM as Pb(NO 3) 2. The root compartment (RC) was isolated from the hyphal compartment (HC) by nylon mesh (37 mm). The glomalin was extracted from both RC and HC and assayed by using Bradford method and monoclonal antibody MAb32B11. The amount of sequestrated Pb was determined after wet digestion of glomalin. The results showed that the amount of Bradford reactive and immunoreactive glomalin is not correlated with hyphal biomass at different Pb levels. There was a positive and significant relationship between the percentage of root colonization and both immunoreactive and Bradford reactive glomalin. The content of Bradford and immunoreactive glomalin in both RC and HC was significantly increased by rising Pb levels, compared to the unleaded control. The maximum immuno-reactive glomalin in HC (10.04 mg/mg hyphae) and in RC (7.79 mg/mg root) was recorded at 450 mM of Pb, which was markedly different from other levels of Pb. Total Pb sequestrated by HC glomalin enhanced as the level of Pb increased and it was 665.72 mg Pb/mg HC glomalin at 450 mM of Pb, while the total Pb sequestrated by RC glomalin was enhanced as the levels of Pb increased up to 300 mM (228.91 mg Pb/mg RC glomalin). Also, there were positive and significant correlations between both immunoreactive and Bradford reactive glomalin in HC with the amount of Pb sequestration. Consequently, this study provides evidence on the role of glomalin in Pb sequestration which should be considered for biostabilization of toxic elements in polluted sites.
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Glomalin (a glycoprotein) is produced by arbuscular mycorrhizal fungi and has the ability to sequester toxic heavy metals. We hypothesized that an increase in the concentration of Pb would lead to increased glomalin production and... more
Glomalin (a glycoprotein) is produced by arbuscular mycorrhizal fungi and has the ability to sequester toxic heavy metals. We hypothesized that an increase in the concentration of Pb would lead to increased glomalin production and consequently higher Pb-sequestration. In a two compartment pot culture experiment, clover plants (Trifolium repens L.) were inoculated with arbuscular mycorrhizal fungus, Rhizophagus irregularis, in sterile washed sand and then treated with Pb concentrations of 0, 150, 300 and 450 mM as Pb(NO 3) 2. The root compartment (RC) was isolated from the hyphal compartment (HC) by nylon mesh (37 mm). The glomalin was extracted from both RC and HC and assayed by using Bradford method and monoclonal antibody MAb32B11. The amount of sequestrated Pb was determined after wet digestion of glomalin. The results showed that the amount of Bradford reactive and immunoreactive glomalin is not correlated with hyphal biomass at different Pb levels. There was a positive and significant relationship between the percentage of root colonization and both immunoreactive and Bradford reactive glomalin. The content of Bradford and immunoreactive glomalin in both RC and HC was significantly increased by rising Pb levels, compared to the unleaded control. The maximum immuno-reactive glomalin in HC (10.04 mg/mg hyphae) and in RC (7.79 mg/mg root) was recorded at 450 mM of Pb, which was markedly different from other levels of Pb. Total Pb sequestrated by HC glomalin enhanced as the level of Pb increased and it was 665.72 mg Pb/mg HC glomalin at 450 mM of Pb, while the total Pb sequestrated by RC glomalin was enhanced as the levels of Pb increased up to 300 mM (228.91 mg Pb/mg RC glomalin). Also, there were positive and significant correlations between both immunoreactive and Bradford reactive glomalin in HC with the amount of Pb sequestration. Consequently, this study provides evidence on the role of glomalin in Pb sequestration which should be considered for biostabilization of toxic elements in polluted sites.
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The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of... more
The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against tumor necrosis factor-alpha (TNF- α), which is a potent pro-inflammatory cytokine and plays important role in various inflammatory diseases and malignancies. To pursue production of scFv antibody fragments against human TNF- α, we performed five rounds of biopanning using stepwise decreased amount of TNF-α (1 to 0.1 μ g), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR, restriction fragment length polymorphism (RFLP) and Western blot analyses as well as fluorescence microscopy and flow cytometry. Based upon binding affinity to TNF-α , 15 clones were selected out of 50 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human TNF-α . The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the Raji B lymphoblasts. The effectiveness of the selected scFv fragments was further validated by flow cytometry analysis in the lipopolysaccharide (LPS) treated mouse fibroblast L929 cells. Based upon these findings, we propose the selected fully human anti-TNF-α scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications.
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Using phage display technology (PDT), we have recently isolated single-chain variable fragment (scFv) antibodies (Ab) against some pivotal molecular markers involved in malignancies and systemic inflammation including human tumor necrosis... more
Using phage display technology (PDT), we have recently isolated single-chain variable fragment (scFv) antibodies (Ab) against some pivotal molecular markers involved in malignancies and systemic inflammation including human tumor necrosis factor-alpha (TNF-α, GI:367465798). Downstream purification and characterization of scFv antibodies is a challenging issue, which may impose substantial impacts on quality of the final product(s). Of various purification methods, affinity chromatography has widely been used in the pharmaceutical grade downstream processing (PGDP) of proteins (e.g., monoclonal antibody (mAb) and scFvs). To pursue an optimized PGDP, in the current study, we have capitalized PDT for upstream selection of anti-TNF-α scFvs and protein A affinity chromatography (PAAC) for downstream extraction and purification of the scFvs from the crude medium secreted and periplasmic fractions of HB2151 cells. The versatility of the PDT selection was validated using SDS-PAGE electrophoresis, western blot, dot blot, ELISA and fluorescence microscopy. SDS-PAGE western blot analyses displayed a 17 kDa scFv with high purity (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 98%), while dot blot and ELISA analyses showed high specificity and binding affinity of the purified scFv antibody fragments toward human TNF-α at nM range. Fluorescence microscopy further confirmed detection of TNF-α in Raji B lymphoblast cells. Finally, based on our findings, we believe that these PDT selected and PAAC processed scFvs may be used as targeting and/or therapy agent for TNF-α mediated diseases. Further, to increase the production yield, downstream purification techniques need to be optimized for the large scale production of scFv antibodies.
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Research Interests: Molecular Biology, Biology, CD, Antibody, Monoclonal Antibody, and 2 morePeritoneum and Antigen(Peritoneum and Antigen)
(Peritoneum and Antigen)
Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with... more
Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases.
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Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of... more
Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs) with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells.
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Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34... more
Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells.
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Polyclonal antibodies against kappa light chain are used to diagnose diseases producing free light chain. The kappa and lambda light chains are products of immunoglobulin synthesis and released into the circulation in minor amounts such... more
Polyclonal antibodies against kappa light chain are used to diagnose diseases producing free light chain. The kappa and lambda light chains are products of immunoglobulin synthesis and released into the circulation in minor amounts such as serum, cerebrospinal fluid, urine and synovial fluid in normal condition. The purpose of this study was the production and purification of polyclonal immunoglobulin G (IgG) against human kappa light chains. In this study, early human IgG was purified by ion-exchange chromatography, reduced with Dithiothreitol and heavy and light chains were separated with size-exclusion chromatography. Afterward, affinity chromatography with protein L Sepharose at pH 2.00 was displayed to be a dominant condition for the separation and purification of the kappa light chain of immunoglobulins from human serum. Eventually, the rabbit was immunized by human kappa light chains. The rabbit IgG was purified and labeled with horseradish peroxidase (HRP). Direct enzyme-linked immunosorbent assay was planned to determine the titer of HRP conjugated rabbit IgG against the human kappa light chain. The optimum titer of anti-kappa IgG was 1:16000. At the result, purified polyclonal anti-kappa is useful tool in biomedical and biochemical researches and diagnostic kits.
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As the most predominant protein in plasma, albumin is synthesized in the liver. . Given to various applications of albumin as biopharmaceutical agent, the annual demand for it is 500 tons in the world, which is the highest in the... more
As the most predominant protein in plasma, albumin is synthesized in the liver. . Given to various applications of albumin as biopharmaceutical agent, the annual demand for it is 500 tons in the world, which is the highest in the biomedical solutions demand ranking. There exist different procedures for production of albumin. The aim of this study was the purification of human serum albumin (HSA) using immunoaffinity chromatography. After immunization of rabbits, passive immunodiffusion and indirect ELISA tests were applied for assessment of polyclonal antibody production against HSA. Purification was performed by ion exchange chromatography (IEC) and protein G affinity chromatography. The produced anti-HSA IgG was attached to the CNBR-activated Sepharose and applied for albumin purification from human serum. Western blotting (WB) analysis and heat-induced insolubility were performed for functional and stability measurement assessment of immunoaffinity purified HSA, respectively. The optimum titer of anti-HSA determined by indirect ELISA was 256000. The SDS-PAGE showed that the purity rate of albumin was approximately %98 and WB confirmed the HSA functionality. Also the heat-induced insolubility of immunoaffinity purified HSA was the same as the commercial HSA. Affinity chromatography using produced polyclonal antibody would be a robust method for purification of HSA.
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High-mobility group protein two (HMGA2), a nonhistone nuclear-binding protein and its downregulators; vimentin, matrix metallopeptidase-9 (MMP-9), and E-cadherin are shown to contribute to tumor progression and metastasis. Thus, in this... more
High-mobility group protein two (HMGA2), a nonhistone nuclear-binding protein and its downregulators; vimentin, matrix metallopeptidase-9 (MMP-9), and E-cadherin are shown to contribute to tumor progression and metastasis. Thus, in this study, we checked simultaneous delivery of HMGA-2 siRNA and the anticancer drug doxorubicin to enhance the anticancer treatment effects. For this purpose, we used MTT assay and real-time polymerase chain reaction (RT-PCR). Our results showed that dual delivery of Dox and HMGA-2 siRNA by trimethyl chitosan (TMC) significantly inhibited breast cancer cells growth. Additionally, the delivery of siRNA significantly silenced HMGA-2, vimentin, and MMP9 mRNAs, but led to overexpression of E-cadherin mRNA.
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Immunoglobulin A (IgA) is the second immunoglobulin in human serum. Monoclonal antibodies have many potential uses in diagnosis, treatment and purification. The conjugated monoclonal antibodies against human IgA are utilized in diagnostic... more
Immunoglobulin A (IgA) is the second immunoglobulin in human serum. Monoclonal antibodies have many potential uses in diagnosis, treatment and purification. The conjugated monoclonal antibodies against human IgA are utilized in diagnostic kits. For production of monoclonal antibodies against human Immunoglobulin A, spleen cells of the immune mouse were fused with SP2/0 as myeloma cells and Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened to determine the antibody by ELISA. The appropriate clones were selected for limiting dilution (L.D). Ultimately, appropriate monoclone was injected intraperitoneally into the mouse that has been primed with Pristane. In current study, 175 clones were obtained of which 5 clones had absorbance values of about 3 were selected for limiting dilution. Between these clones, 3-D5monoclone with IgG1 subclass was selected as appropriate one and it was reproduced in FCS free RPMI 1640. For large scale production in invivo, the appropriate clone was implanted in the peritoneum of the Balb/c mouse and its titer was determined, which showed 1/100,000 dilution for ascitic fluid, having no cross reactivity with IgM &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp; IgG. Monoclonal antibody was purified by chromatography, confirmed by SDS-PAGE and then conjugated with enzyme and used for diagnostic kits.
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Targeting CD20 antigen on B-lymphocytes provides good opportunity for management of the target cells in patients with B-cell malignancies. By the advent of hybridoma technology, monoclonal antibodies applications exert extensive changes... more
Targeting CD20 antigen on B-lymphocytes provides good opportunity for management of the target cells in patients with B-cell malignancies. By the advent of hybridoma technology, monoclonal antibodies applications exert extensive changes in medical fields such as diagnosis, treatment and purification. The prim aim of this study was to produce monoclonal antibody against CD20 for exploitation in diagnosis. In this study, Balb/c mice were immunized with two peptides from extracellular domain of CD20. Poly Ethylene Glycol (PEG) fused spleen cells of the most immune mouse with SP2/0 (myeloma cells). Supernatant of hybridoma cells were screened for detection of antibody by ELISA. The desired clones were selected for limiting dilution (L.D). Afterward, specificity and cross reactivity of these antibodies were determined by immunological assay such as ELISA and western blot analysis (WB) and Immunofluorescence. Large scale of monoclonal antibodies was produced by ascetic fluid method. Monoclonal antibody was purified by chromatography then confirmed by SDS-PAGE. In this study, between five positive clone wells, 3 clones were chosen for limiting dilution. Limiting dilution product was one monoclone with absorbance about 2. These results indicate that such monoclonal antibodies against CD20 can be used in diagnosis of CD20 in the cells surface.
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Background: In spite of Bacillus Calmette-Guerin (BCG) vaccination, still tuberculosis caused by Mycobacterium tuberculosis remains a problematic impediment that requires perspective management. The aim of this study was to evaluate the... more
Background: In spite of Bacillus Calmette-Guerin (BCG) vaccination, still tuberculosis caused by Mycobacterium tuberculosis remains a problematic impediment that requires perspective management. The aim of this study was to evaluate the immunogenicity of chitosan nanoparticles containing recombinant mycobacterial proteins and adjuvant in the Balb/C mice through a non-invasive nasal inhalation delivery route and measure the level of cytokines interferon gamma (IFN-γ), interleukin-4 (IL-4), and IL-17. Methods: Thirty mice in five different groups were vaccinated through inhalation with compounds set in different combinations. Two weeks after the last nasal delivery, IFN-γ, IL-4, and IL-17 were measured in spleen cell culture supernatants. Results: The IFN-γ and IL-17 concentrations were found to increase in the groups that received chitosan nanoparticles containing protein and adjuvant alone or as a BCG booster. Our study showed that the chitosan nanoparticle containing protein and ad...
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This study attempted to explore the immunogenicity of chitosan nanoparticles containing fusion protein (Hspx–PPE44–EsxV; HPE) and resiquimod adjuvant (HPERC) in BALB/c mice. HPE was initially expressed in E. coli BL21 cells. HPE and... more
This study attempted to explore the immunogenicity of chitosan nanoparticles containing fusion protein (Hspx–PPE44–EsxV; HPE) and resiquimod adjuvant (HPERC) in BALB/c mice. HPE was initially expressed in E. coli BL21 cells. HPE and resiquimod adjuvant were then encapsulated in chitosan nanoparticles (HPERC). One group of mice were subcutaneously vaccinated on days 0, 14, and 28 with HPERC, and the other group was primed with bacilli Calmette-Guérin (BCG) on day 0 and then boosted with HPERC on days 14 and 28. Two weeks after the last injection, IFN-γ, IL-4, and IL-17 in spleen cell culture supernatants, and IgG2a and IgG1 titers in sera were measured. HPERC size was 130.84 ± 12.08 nm ( n = 5). Zeta potential of HPERC was 29 ± 4 mv. The highest IFN-γ concentration was detected in BCG-primed mice that were boosted with HPERC. In addition, IL-17 production was significantly increased in all groups compared with that of control, except in those that received nanoparticle (NP), adjuvant...
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INTRODUCTION Probiotics are live microorganisms, habituated in the human intestine, which have a beneficial effect on our health. In spite of many reports about the anticancer effect of these bacteria in in-vivo and in-vitro, their... more
INTRODUCTION Probiotics are live microorganisms, habituated in the human intestine, which have a beneficial effect on our health. In spite of many reports about the anticancer effect of these bacteria in in-vivo and in-vitro, their mechanisms of action are not completely understood. The goal of this study was to compare the extracellular fractions of Lactobacillus casei and L. paracasei on the anti proliferation and apoptosis induction in K562 cell line. MATERIALS AND METHODS L. casei and L. paracasei were cultured in MRS broth medium. Then extracellular secretions were collected and after enrichment, analyzed by electrophoresis. Fractionation were determined by gel filtration chromatography using sephadex G100 column, and the anticancer properties were evaluated. RESULTS The results of SDS-PAGE showed various molecular weight of fractionated proteins of L. casei and L. paracasei. Bioactivity assessment illustrated that anti proliferative effects on K562 cells is dose and time depen...
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Background: Lipopolysaccharides (LPSs) and several anti- genic proteins of Brucella have been considered for prepara- tion of diagnostic reagents and subunit vaccines. The objective of this study was to identify and compare immunogens of... more
Background: Lipopolysaccharides (LPSs) and several anti- genic proteins of Brucella have been considered for prepara- tion of diagnostic reagents and subunit vaccines. The objective of this study was to identify and compare immunogens of B. abortus S19 which induce humoral immune responses in hu- man, goat and rabbit. Material and Methods: The bacterial whole cell extract was prepared in extraction
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Western blotting or immunoblotting commonly use for study of reaction between antigens and antibodies. Denaturation of many proteins in immunoblotting can affect greatly the reactivity of antibodies and outcome of the procedure. In this... more
Western blotting or immunoblotting commonly use for study of reaction between antigens and antibodies. Denaturation of many proteins in immunoblotting can affect greatly the reactivity of antibodies and outcome of the procedure. In this study proteins of Brucella abortus (S19) was extracted by a mild method and reaction of the extracted proteins with serum of infected human and goat and immunized rabbit compared by affinity chromatography and immunoblotting. Gamma globulin (mostly IgG) fraction of the sera was precipitated by half saturation of ammonium sulfate and linked to activated sepharose 4B. The extracted proteins were loaded on the affinity column. Attached proteins was eluted by low pH and analyzed by SDS-PAGE. Reaction of the total extract and eluted fractions with IgG fraction of sera was evaluated by Western blotting. Upon the results of affinity chromatography and immunoblotting , Brucella proteins can be classified in four groups: 1- The proteins that adsorbed to the affinity column and react with IgG in westernblotting. 2- Proteins that react with IgG in native state but no in denatured state. 3- Proteins that do not react with IgG in native state but react in denatured state. 4- Proteins that do not react with IgG in native and denatured state.
Research Interests: Antibody()
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Cancer stem cells (CSCs) with their self-renewal ability are accepted as cells which initiate tumors. CSCs are regarded as interesting targets for novel anticancer therapeutic agents because of their association with tumor recurrence and... more
Cancer stem cells (CSCs) with their self-renewal ability are accepted as cells which initiate tumors. CSCs are regarded as interesting targets for novel anticancer therapeutic agents because of their association with tumor recurrence and resistance to conventional therapies, including radiotherapy and chemotherapy. Chimeric antigen receptor (CAR)-T cells are engineered T cells which express an artificial receptor specific for tumor associated antigens (TAAs) by which they accurately target and kill cancer cells. In recent years, CAR-T cell therapy has shown more efficiency in cancer treatment, particularly regarding blood cancers. The expression of specific markers such as TAAs on CSCs in varied cancer types makes them as potent tools for CAR-T cell therapy. Here we review the CSC markers that have been previously targeted with CAR-T cells, as well as the CSC markers that may be used as possible targets for CAR-T cell therapy in the future. Furthermore, we will detail the most important obstacles against CAR-T cell therapy and suggest solutions.
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Research Interests: Biology, Medicine, Gene expression, Biological Sciences, Antibodies, and 15 moreCercopithecus aethiops, Environmental Sciences, FISH, Escherichia coli, Animals, Polycyclic Aromatic Hydrocarbon, Aryl Hydrocarbon Receptor, Protein Expression, Rabbits, Sensitivity and Specificity, Recombinant Protein, Recombinant Proteins, Aromatic Hydrocarbon, Signaling pathway, and Medical and Health Sciences(Cercopithecus aethiops, Environmental Sciences, FISH, Escherichia coli, Animals, Polycyclic Aromatic Hydrocarbon, Aryl Hydrocarbon Receptor, Protein Expression, Rabbits, Sensitivity and Specificity, Recombinant Protein, Recombinant Proteins, Aromatic Hydrocarbon, Signaling pathway, and Medical and Health Sciences)
(Cercopithecus aethiops, Environmental Sciences, FISH, Escherichia coli, Animals, Polycyclic Aromatic Hydrocarbon, Aryl Hydrocarbon Receptor, Protein Expression, Rabbits, Sensitivity and Specificity, Recombinant Protein, Recombinant Proteins, Aromatic Hydrocarbon, Signaling pathway, and Medical and Health Sciences)
Nowadays, by advent of hybridoma technology in monoclonal antibodies production various cell markers could be evaluated in malignant and non-malignant cells. CD20, nonglycosylated phosphoprotein is as an ideal marker in leukemia and... more
Nowadays, by advent of hybridoma technology in monoclonal antibodies production various cell markers could be evaluated in malignant and non-malignant cells. CD20, nonglycosylated phosphoprotein is as an ideal marker in leukemia and B-cell lymphoma diagnosis which is expressed on more than 95% of normal and neoplastic B-cells except for early B-cells and mature plasma. The prime aim of this study was to produce monoclonal antibody against CD20 and evaluation of specific binding to CD20 expressing cells. Materials & Methods: First, Balb/c mice were immunized 4 times with 100µg peptide from extracellular domain of CD20. Spleen cells of the most immune mouse were fused with SP2/0 by Poly Ethylene Glycol (PEG). The desired clones were selected for limiting dilution (L.D). Large scale of monoclonal antibodies was produced by ascetic fluid method. Monoclonal antibody was purified by Protein-A-Sepharose column affinity chromatography then confirmed by SDS-PAGE. Afterward, evaluation of spe...
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BACKGROUND Republic of Azerbaijan is considered as an area with high prevalence of multi-drug resistant tuberculosis. Uncontrolled travelling of Azerbaijanis people to Iran is the issue that needs to be considered as an important issue.... more
BACKGROUND Republic of Azerbaijan is considered as an area with high prevalence of multi-drug resistant tuberculosis. Uncontrolled travelling of Azerbaijanis people to Iran is the issue that needs to be considered as an important issue. METHODS This study was conducted on 32 patients with tuberculosis from Baku-Nakhchivan and 48 patients from Iran during 2012 to 2014. Colonies of Mycobacterium tuberculosis were examined after isolating them from patients using proportional method on Lowenstein-Jensen media regarding resistance encounter with Rifampin, Isoniazid and Ethambutol. RESULTS Among M. tuberculosis isolates belonging to 32 foreign patients; 69%, 72% and 56% of them were resistant to Rifampin, Isoniazid and Ethambutol, respectively (multidrug resistance tuberculosis: MDR-TB: 62.5%). From 48 isolates of Iranian patients; 8%, 4% and 4% were resistant to Rifampin, Isoniazid and Ethambutol, respectively (MDR-TB: 2.1%). CONCLUSIONS Resistant strains are common in Baku-Nakhchivan&#...
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Apelin is an endogenous peptide, which is expressed in a vast board of organs such as the brain, placenta, heart, lungs, kidneys, pancreas, testis, prostate and adipose tissues. The apelin receptor, called angiotensin-like-receptor 1... more
Apelin is an endogenous peptide, which is expressed in a vast board of organs such as the brain, placenta, heart, lungs, kidneys, pancreas, testis, prostate and adipose tissues. The apelin receptor, called angiotensin-like-receptor 1 (APJ), is also expressed in the brain, spleen, placenta, heart, liver, intestine, prostate, thymus, testis, ovary, lungs, kidneys, stomach, and adipose tissue. The apelin/APJ axis is involved in a number of physiological and pathological processes. The apelin expression is increased in various kinds of cancer and the apelin/APJ axis plays a key role in the development of tumors through enhancing angiogenesis, metastasis, cell proliferation and also through the development of cancer stem cells and drug resistance. The apelin also stops the apoptosis of cancer cells. The apelin/APJ axis was considered in this review as an attractive therapeutic target for cancer treatment.
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Abstract Glomalin (a glycoprotein) is produced by arbuscular mycorrhizal fungi and has the ability to sequester toxic heavy metals. We hypothesized that an increase in the concentration of Pb would lead to increased glomalin production... more
Abstract Glomalin (a glycoprotein) is produced by arbuscular mycorrhizal fungi and has the ability to sequester toxic heavy metals. We hypothesized that an increase in the concentration of Pb would lead to increased glomalin production and consequently higher Pb-sequestration. In a two compartment pot culture experiment, clover plants (Trifolium repens L.) were inoculated with arbuscular mycorrhizal fungus, Rhizophagus irregularis , in sterile washed sand and then treated with Pb concentrations of 0, 150, 300 and 450 μM as Pb(NO 3 ) 2 . The root compartment (RC) was isolated from the hyphal compartment (HC) by nylon mesh (37 μm). The glomalin was extracted from both RC and HC and assayed by using Bradford method and monoclonal antibody MAb32B11. The amount of sequestrated Pb was determined after wet digestion of glomalin. The results showed that the amount of Bradford reactive and immunoreactive glomalin is not correlated with hyphal biomass at different Pb levels. There was a positive and significant relationship between the percentage of root colonization and both immunoreactive and Bradford reactive glomalin. The content of Bradford and immunoreactive glomalin in both RC and HC was significantly increased by rising Pb levels, compared to the unleaded control. The maximum immunoreactive glomalin in HC (10.04 μg/mg hyphae) and in RC (7.79 μg/mg root) was recorded at 450 μM of Pb, which was markedly different from other levels of Pb. Total Pb sequestrated by HC glomalin enhanced as the level of Pb increased and it was 665.72 μg Pb/mg HC glomalin at 450 μM of Pb, while the total Pb sequestrated by RC glomalin was enhanced as the levels of Pb increased up to 300 μM (228.91 μg Pb/mg RC glomalin). Also, there were positive and significant correlations between both immunoreactive and Bradford reactive glomalin in HC with the amount of Pb sequestration. Consequently, this study provides evidence on the role of glomalin in Pb sequestration which should be considered for biostabilization of toxic elements in polluted sites.
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Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein... more
Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. In this study, we examined the potential of our produced anti-TNF-α scFv fragments for purification of TNF-α produced by Raji cells. The Raji cells were induced by lipopolysaccharides (LPS) to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Western blot and FACS flow cytometry analyses showed the successful expression of ...
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Research Interests: Environmental Toxicology, Flow Cytometry, Biology, Cell Cycle, Apoptosis, and 15 moreMedicine, Humans, Alkaloids, Caspase, Protein Expression, Cell Proliferation, Growth Factor, Cell Survival, Enzyme Linked Immunosorbent Assay, Control Group, ENVIRONMENTAL SCIENCE AND MANAGEMENT, DNA fragmentation, reverse transcriptase polymerase chain reaction, Reverse transcription polymerase chain reaction, and Pharmacology and pharmaceutical sciences(Medicine, Humans, Alkaloids, Caspase, Protein Expression, Cell Proliferation, Growth Factor, Cell Survival, Enzyme Linked Immunosorbent Assay, Control Group, ENVIRONMENTAL SCIENCE AND MANAGEMENT, DNA fragmentation, reverse transcriptase polymerase chain reaction, Reverse transcription polymerase chain reaction, and Pharmacology and pharmaceutical sciences)
(Medicine, Humans, Alkaloids, Caspase, Protein Expression, Cell Proliferation, Growth Factor, Cell Survival, Enzyme Linked Immunosorbent Assay, Control Group, ENVIRONMENTAL SCIENCE AND MANAGEMENT, DNA fragmentation, reverse transcriptase polymerase chain reaction, Reverse transcription polymerase chain reaction, and Pharmacology and pharmaceutical sciences)