- Email: [email protected]
Production and characterization of a polyclonal antibody against an actin peptide
Abstracts
Oral – [A-10-819-1] Effect of ω-3 and ω-6 polyunsaturated fatty acids administration on secretory phospholipase A2 type IIa level and cell survival in ectopic and eutopic endometrial cell cultures in patients with endometriosis Korosh Khanakia, Mohammad Nourib, Ali Rahimipourc, Ali M. Ardekanid, Mohammad R. Sadeghid, Alieh Ghassemzadehb, Vahideh Shahnazib, R. Imani Alie, Abotaleb Saremif, Masoud Darabia a Department of Clinical Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran b Women's Reproductive Health Research Center, Alzahra Hospital, Tabriz, Iran c Shahid Beheshti University of Medical Sciences, Tehran, Iran d Avicenna Research Institute, Tehran, Iran e Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran f Sarem Hospital, Tehran, Iran E-mail addresses: [email protected] (K. Khanaki), [email protected] (M. Nouri), [email protected] (A.M. Ardekani), [email protected] (A. Ghassemzadeh), [email protected] (V. Shahnazi), [email protected] (R. Imani Ali), [email protected] (M. Darabi) Introduction: The etiology of Endometriosis, a common gynecological disease, is highly linked to chronic inflammation. Secretory phospholipase A2 type IIa (sPLA2IIa) is an acute phase reactant that is markedly increased in inflammatory disorders. We evaluated the effect of supplementation of ω-3 and ω-6 PUFAs in culture medium on sPLA2IIa level and cell survival in ectopic versus eutopic endometrial cell cultures from endometriosis patients. Methods: In vitro cell culture study of paired ectopic and eutopic endometrial tissue sampled from fifteen endometriosis patients. Cells were cultured for 8 days in three different culture media; balanced ω3/ω-6, high ω-3 and high ω-6 PUFAs ratio. Cell survival was measured using 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino) carbonyl-2H-tetrazolium hydroxide (XTT) method and sPLA2IIa level assessed with ELISA technique. Comparisons of the three different fatty acid treatments were performed between paired ectopic versus eutopic endometrial cell cultures and within each of the two types of endometriotic tissue cell groups. Results: The sPLA2IIa level was significantly higher in the ectopic endometrial cell culture compared to the eutopic group for each of the three matched treatments (balanced, high ω-3 and high ω-6). Cell survival in the eutopic group was significantly decreased by high ω-6 culturing compared to control medium. Conclusion: The increase in sPLA2IIa level in ectopic endometrial cells by fatty acid treatments (especially high ω-3), strengthens the hypothesis that PUFAs stimulate cytokines increasing sPLA2IIa level. Further studies elucidating sPLA2IIa, and PUFAs role in the disease process and treatment of endometriosis would be welcomed. Keywords: Fatty acids, Endometriosis, Secretory phospholipase A2 type IIa, Cell culture doi:10.1016/j.clinbiochem.2011.08.016
E.poster – [A-10-916-2] Immune response profile in estrogen treated ovariectomized experimental autoimmune encephalomyelitis induced mice Abbas Ali Amini, Dariush Haghmorad, Mitra Masoudian, Shahrzad Zamani, Nafise Tabasi, Maryam Rastin, Mahmoud Mahmoudi Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran E-mail addresses: [email protected] (A.A. Amini), [email protected] (D. Haghmorad), [email protected] (M. Mahmoudi)
S5
Introduction: Multiple sclerosis is the most common inflammatory disease in the central nervous system, which is due to the reaction of auto reactive T cells with myelin proteins, leading to physical disorder and paralysis. Experimental autoimmune encephalomyelitis (EAE) is used as an animal model of this disease. Materials and method: Twenty four C57/BL6 female mice, aging 8– 10 weeks and weighing 20 gr were used in this project. The mice were divided into 3 groups as follows: 1. Normal group 2. Control ovariectomized group 3. Estrogen (E2) treated ovariectomized group. For induction of EAE a mixture of Myelin oligodendrocyte Glycoprotein and complete freund's adjuvant were injected subcutaneously. Mice were also intraperitoneally (i.p.) injected with pertussis toxin. A second identical injection of pertussis toxin was given after 48 h. Parallel to EAE induction, the treatment group twice a week for 21 days was injected i.p 2 mg/kg estrogen. On 21 day, mice were anesthetized and sacrificed. Percents of FoxP3+ regulatory splenocytes by flow cytometry, levels of IL-4, IL-17 and IFN-g using ELISA and splenocytes proliferation assay by Brdu, were all covered to determine the profile of immune response. Results: Despite decreased in the rate of FoxP3+ regulatory T cells and IL-4 secretion, the levels of IFN-g, IL-17 and the proliferation of splenocytes all showed remarkable increase in E2-treated ovariectomized group. Conclusion: This study suggests that estrogen shifted the immune response to Th1 and Th17. Keywords: Multiple sclerosis, Experimental autoimmune encephalomyelitis, Estrogen, Ovariecromized doi:10.1016/j.clinbiochem.2011.08.017
Oral – [A-10-922-1] Production and characterization of a polyclonal antibody against an actin peptide Nazila Amini, Elham Safarpur, Reza Hadavi, Hodjattallah Rabbani, Ahmad-Reza Mahmoudi, Ali-Ahmad Bayat, Jafar Mahmoudian, Mohaddeseh Naghi-Vishteh, Jeddi-Tehrani Mahmood Department of Biology, Science and research branch, Islmic Azad University, Tehran, Iran E-mail address: [email protected] (N. Amini) Introduction: Actin is the major cytoskeletal protein in all eukaryotic cells. Chemically, small differences in amino acid content exist in actin from different species. However, these differences apparently do not influence the antigenicity. The level of expression of actin is essentially the same in different kinds of cells. The aim of this project was to produce a polyclonal antibody against an actin peptide. Materials and methods: A synthetic peptide from actin was designed, produced and conjugated to KLH and used for immunization of a New Zealand White rabbit. An affinity column was prepared by coupling the immunizing peptide to CN-Br-activated sepharose 4B. The produced antibody was purified by affinity chromatography and its specific interaction with the immunizing peptide was determined by ELISA. Cell lysate was prepared from Hela cells and then subjected to Western blot. The reactivity of the antibody with actin protein was also tested by immunocytochemical staining of Hela cells. Result and conclusion: The antibody showed strong reactivity with the immunizing peptide in ELISA and could detect a single 43 kDa band in Western blot. Hela cells were also stained strongly by the antibody in immunocytochemistry. The antibody may be used as a tool for diagnostic purposes and also for normalization of protein load in biological samples. Keywords: Polyclonal antibody, Western blotting, Immunocytochemistry, Actin doi:10.1016/j.clinbiochem.2011.08.018
Oral – [A-10-819-1] Effect of ω-3 and ω-6 polyunsaturated fatty acids administration on secretory phospholipase A2 type IIa level and cell survival in ectopic and eutopic endometrial cell cultures in patients with endometriosis Korosh Khanakia, Mohammad Nourib, Ali Rahimipourc, Ali M. Ardekanid, Mohammad R. Sadeghid, Alieh Ghassemzadehb, Vahideh Shahnazib, R. Imani Alie, Abotaleb Saremif, Masoud Darabia a Department of Clinical Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran b Women's Reproductive Health Research Center, Alzahra Hospital, Tabriz, Iran c Shahid Beheshti University of Medical Sciences, Tehran, Iran d Avicenna Research Institute, Tehran, Iran e Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran f Sarem Hospital, Tehran, Iran E-mail addresses: [email protected] (K. Khanaki), [email protected] (M. Nouri), [email protected] (A.M. Ardekani), [email protected] (A. Ghassemzadeh), [email protected] (V. Shahnazi), [email protected] (R. Imani Ali), [email protected] (M. Darabi) Introduction: The etiology of Endometriosis, a common gynecological disease, is highly linked to chronic inflammation. Secretory phospholipase A2 type IIa (sPLA2IIa) is an acute phase reactant that is markedly increased in inflammatory disorders. We evaluated the effect of supplementation of ω-3 and ω-6 PUFAs in culture medium on sPLA2IIa level and cell survival in ectopic versus eutopic endometrial cell cultures from endometriosis patients. Methods: In vitro cell culture study of paired ectopic and eutopic endometrial tissue sampled from fifteen endometriosis patients. Cells were cultured for 8 days in three different culture media; balanced ω3/ω-6, high ω-3 and high ω-6 PUFAs ratio. Cell survival was measured using 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino) carbonyl-2H-tetrazolium hydroxide (XTT) method and sPLA2IIa level assessed with ELISA technique. Comparisons of the three different fatty acid treatments were performed between paired ectopic versus eutopic endometrial cell cultures and within each of the two types of endometriotic tissue cell groups. Results: The sPLA2IIa level was significantly higher in the ectopic endometrial cell culture compared to the eutopic group for each of the three matched treatments (balanced, high ω-3 and high ω-6). Cell survival in the eutopic group was significantly decreased by high ω-6 culturing compared to control medium. Conclusion: The increase in sPLA2IIa level in ectopic endometrial cells by fatty acid treatments (especially high ω-3), strengthens the hypothesis that PUFAs stimulate cytokines increasing sPLA2IIa level. Further studies elucidating sPLA2IIa, and PUFAs role in the disease process and treatment of endometriosis would be welcomed. Keywords: Fatty acids, Endometriosis, Secretory phospholipase A2 type IIa, Cell culture doi:10.1016/j.clinbiochem.2011.08.016
E.poster – [A-10-916-2] Immune response profile in estrogen treated ovariectomized experimental autoimmune encephalomyelitis induced mice Abbas Ali Amini, Dariush Haghmorad, Mitra Masoudian, Shahrzad Zamani, Nafise Tabasi, Maryam Rastin, Mahmoud Mahmoudi Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran E-mail addresses: [email protected] (A.A. Amini), [email protected] (D. Haghmorad), [email protected] (M. Mahmoudi)
S5
Introduction: Multiple sclerosis is the most common inflammatory disease in the central nervous system, which is due to the reaction of auto reactive T cells with myelin proteins, leading to physical disorder and paralysis. Experimental autoimmune encephalomyelitis (EAE) is used as an animal model of this disease. Materials and method: Twenty four C57/BL6 female mice, aging 8– 10 weeks and weighing 20 gr were used in this project. The mice were divided into 3 groups as follows: 1. Normal group 2. Control ovariectomized group 3. Estrogen (E2) treated ovariectomized group. For induction of EAE a mixture of Myelin oligodendrocyte Glycoprotein and complete freund's adjuvant were injected subcutaneously. Mice were also intraperitoneally (i.p.) injected with pertussis toxin. A second identical injection of pertussis toxin was given after 48 h. Parallel to EAE induction, the treatment group twice a week for 21 days was injected i.p 2 mg/kg estrogen. On 21 day, mice were anesthetized and sacrificed. Percents of FoxP3+ regulatory splenocytes by flow cytometry, levels of IL-4, IL-17 and IFN-g using ELISA and splenocytes proliferation assay by Brdu, were all covered to determine the profile of immune response. Results: Despite decreased in the rate of FoxP3+ regulatory T cells and IL-4 secretion, the levels of IFN-g, IL-17 and the proliferation of splenocytes all showed remarkable increase in E2-treated ovariectomized group. Conclusion: This study suggests that estrogen shifted the immune response to Th1 and Th17. Keywords: Multiple sclerosis, Experimental autoimmune encephalomyelitis, Estrogen, Ovariecromized doi:10.1016/j.clinbiochem.2011.08.017
Oral – [A-10-922-1] Production and characterization of a polyclonal antibody against an actin peptide Nazila Amini, Elham Safarpur, Reza Hadavi, Hodjattallah Rabbani, Ahmad-Reza Mahmoudi, Ali-Ahmad Bayat, Jafar Mahmoudian, Mohaddeseh Naghi-Vishteh, Jeddi-Tehrani Mahmood Department of Biology, Science and research branch, Islmic Azad University, Tehran, Iran E-mail address: [email protected] (N. Amini) Introduction: Actin is the major cytoskeletal protein in all eukaryotic cells. Chemically, small differences in amino acid content exist in actin from different species. However, these differences apparently do not influence the antigenicity. The level of expression of actin is essentially the same in different kinds of cells. The aim of this project was to produce a polyclonal antibody against an actin peptide. Materials and methods: A synthetic peptide from actin was designed, produced and conjugated to KLH and used for immunization of a New Zealand White rabbit. An affinity column was prepared by coupling the immunizing peptide to CN-Br-activated sepharose 4B. The produced antibody was purified by affinity chromatography and its specific interaction with the immunizing peptide was determined by ELISA. Cell lysate was prepared from Hela cells and then subjected to Western blot. The reactivity of the antibody with actin protein was also tested by immunocytochemical staining of Hela cells. Result and conclusion: The antibody showed strong reactivity with the immunizing peptide in ELISA and could detect a single 43 kDa band in Western blot. Hela cells were also stained strongly by the antibody in immunocytochemistry. The antibody may be used as a tool for diagnostic purposes and also for normalization of protein load in biological samples. Keywords: Polyclonal antibody, Western blotting, Immunocytochemistry, Actin doi:10.1016/j.clinbiochem.2011.08.018