Masoud Hassanzadeh Makoui; Maryam Mobini; Jalal Khoshnoodi; Tannaz Bahadori; Forough Golsaz-Shirazi; Hedieh Moradi Tabriz; Zahra Madjd; Mahmood Jeddi-Tehrani; Amir-Hassan Zarnani; Mohammad Mehdi Amiri; Fazel Shokri
Abstract
Background: Ki67 and P53 are important diagnostic and prognostic biomarkers expressed in several cancers. The current standard method for evaluating Ki67 and P53 in cancer tissues is immunohistochemistry (IHC), and having highly sensitive monoclonal antibodies against these biomarkers is necessary for ...
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Background: Ki67 and P53 are important diagnostic and prognostic biomarkers expressed in several cancers. The current standard method for evaluating Ki67 and P53 in cancer tissues is immunohistochemistry (IHC), and having highly sensitive monoclonal antibodies against these biomarkers is necessary for an accurate diagnosis in the IHC test.Objective: To generate and characterize novel monoclonal antibodies (mAbs) against human Ki67 and P53 antigens for IHC purposes.Methods: Ki67 and P53-specific mAbs were produced by the hybridoma method and screened by enzyme-linked immunosorbent assay (ELISA) and IHC techniques. Selected mAbs were characterized using Western blot and flow cytometry, and their affinities and isotypes were determined by ELISA. Moreover, using the IHC technique in 200 breast cancer tissue samples, we assessed the specificity, sensitivity, and accuracy of the produced mAbs.Results: Two anti-Ki67 (2C2 and 2H1) and three anti-P53 mAbs (2A6, 2G4, and 1G10) showed strong reactivity to their target antigens in IHC. The selected mAbs were also able to recognize their targets by flow cytometry as well as Western blotting using human tumor cell lines expressing these antigens. The specificity, sensitivity, and accuracy calculated for clone 2H1 were 94.2%, 99.0%, and 96.6%, and for clone 2A6 were 97.3%, 98.1%, and 97.5%, respectively. Using these two monoclonal antibodies, we found a significant correlation between Ki67 and P53 overexpression and lymph node metastasis in patients with breast cancer.Conclusion: The present study showed that the novel anti-Ki67 and anti-P53 mAbs could recognize their respective antigens with high specificity and sensitivity and therefore can be used in prognostic studies.
Fang Gao; Yuan Chen; Wenwei Zhang; Xiaolong Wang; Jingchun Li; Feng Feng; Chunli Liu; Wenxi He; Wei Sun
Abstract
Background: Suppressor APC domain containing 2 (SAPCD2) is involved in cell cycle regulation and its mRNA levels are higher in cancer tissues. But, the function of SAPCD2 in cancer development remains unclear.Objective: To generate mouse monoclonal antibodies (mAbs) specific to SAPCD2 and thus clarify ...
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Background: Suppressor APC domain containing 2 (SAPCD2) is involved in cell cycle regulation and its mRNA levels are higher in cancer tissues. But, the function of SAPCD2 in cancer development remains unclear.Objective: To generate mouse monoclonal antibodies (mAbs) specific to SAPCD2 and thus clarify the function of SAPCD2 in the development of gastric carcinoma (GC).Methods: Purified SAPCD2-GST immunized BALB/c mouse spleen cells were collected and fused with myeloma cells to obtain monoclonal antibody hybridoma. A group of monoclonal antibodies exhibiting high specificity and sensitivity against SAPCD2 has been generated and characterized by IHC, WB, IP, IF, and ELISA. By immunohistochemical analysis, the SAPCD2 expression was evaluated in 228 clinical samples of gastric mucosal lesions, including precancerous lesions and GC samples.Results: We identified a highly specific and sensitive clone of s12 in eukaryotic cells and performed an IHC analysis. We found that 81.3% of 107 GC tissues were SAPCD2-positive, compared with the 26.2% in the matched adjacent normal-appearing tissues (P<0.001). Furthermore, among the 121 gastritis tissues, SAPCD2 was overexpressed in precancerous gastric lesions such as dysplasia (Dys, 78.9%), intestinal metaplasia (IM, 44.7%), and chronic atrophic gastritis (CAG, 6.1%) compared with that in chronic non-atrophic gastritis (CNAG, 3.2%) (P<0.001). The SAPCD2-positivity rate was 81.3% in GC, suggesting that the expression of SAPCD2 increased with the severity of the lesion (P<0.001).Conclusion: In summary, we have described novel monoclonal antibodies against SAPCD2, which are highly expressed in GC tissues and may serve as the basis for an early clinical marker for GC development.
Lia Farahi; Fatemeh Ghaemimanesh; Saeideh Milani; Seyed Mohsen Razavi; Reza Hadavi; Ali Ahmad Bayat; Ali Salimi; Mohammad Mehdi Akhondi; Hodjattallah Rabbani
Abstract
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 ...
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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.
Mahmood Shams; Mahmood Jeddi-Tehrani; Farzaneh Notash Haghighat; Ali Ahmad Bayat; Jafar Mahmoudian; Mohammad Reza Rezvani
Volume 10, Issue 4 , December 2013, , Pages 259-266
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 ...
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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.
Forough Golsaz Shirazi; Mohammad Mehdi Amiri; Hamed Mohammadi; Ali Ahmad Bayat; Azam Roohi; Jalal Khoshnoodi; Amir Hassan Zarnani; Mahmood Jeddi-Tehrani; Gholam Ali Kardar; Fazel Shokri
Volume 10, Issue 3 , September 2013, , Pages 127-138
Abstract
Background: The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect ...
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Background: The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays. Objectives: To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs). Methods: Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA. Results: Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ‘‘a’’ determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs. Conclusion: Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.
Sorour Shojaeian; Amir Hassan Zamani; Mahmood Jeddi-Tehrani; Forouzandeh Fereidooni; Ebrahim Torkabadi; Mohammad Mehdi Akhondi; Akram Sadat Tabatabaei-Panah; Abdolamir Allameh
Volume 6, Issue 4 , December 2009, , Pages 174-185
Abstract
Background: Monoclonal antibodies (mAbs) are essential tools for many molecular im-munology investigations, epitope mapping and molecular modelling, clinical laboratory di-agnostic tests and immunotherapy. Humoral immune response of immunized animals largely depends on the nature of antigen and the immunization ...
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Background: Monoclonal antibodies (mAbs) are essential tools for many molecular im-munology investigations, epitope mapping and molecular modelling, clinical laboratory di-agnostic tests and immunotherapy. Humoral immune response of immunized animals largely depends on the nature of antigen and the immunization technique. Polysaccharides and heavily-glycosylated proteins are very elusive targets incapable of mounting long-lasting, high affinity antibody responses. Carcinoma antigen 125 (CA 125), a well known tumor marker of ovarian cancer, is a mucin type antigen consisting of repetitive units of heavily glycosylated moieties which render production of mAbs very difficult. Objective: To evaluate the efficacy of heterologous antigen preparations as a way of mouse immuniza-tion in the production of anti-CA 125 mAb. Methods: Two different protocols of immuni-zation were used for priming of NMRI mice. In the first method, mice conventionally im-munized by three intraperitoneal injections of purified CA 125 and boosted by the antigen three days before fusion. In the second approach, mice were primed by three intraperitoneal injections of living CA 125 positive cells of OVCAR-3 cell line, and boosted by intrave-nous injection of the purified extracellular domain of CA 125. Production of mAb was per-formed by standard hybridoma technology and mAbs were characterized by different im-munoassays. Results: The first method failed to produce stable clones despite six time fu-sion. A total of ten stable clones, however, were produced in the second approach. Some of the clones were characterized and found to have excellent immunoreactivity when tested by ELISA assay, western blotting, intracellular and surface immunofluorescent staining of OVCAR-3 cell line and immunohistochemical staining of ovarian cancer tissues. Conclusion: Altogether the results of the present study clearly showed that heterologous antigen preparation is the method of choice for immunization when production of mono-clonal antibody against highly glycosylated poorly immunogenic antigens is concerned.
Mojtaba Sankian; Forough Glosaze Shirazi; Mahid Arafi; Malihe Moghadam; Abdolreza Varasteh
Volume 5, Issue 3 , September 2008, , Pages 156-162
Abstract
Background: Allergy to Saffron (Crocus sativus) pollen has been described in people involved in processing of saffron flower stamens. Profilins have been identified as a pan-allergen in different plant pollens and foods. This molecule is an actin-binding pro-tein with a molecular weight of 12-16 kDa ...
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Background: Allergy to Saffron (Crocus sativus) pollen has been described in people involved in processing of saffron flower stamens. Profilins have been identified as a pan-allergen in different plant pollens and foods. This molecule is an actin-binding pro-tein with a molecular weight of 12-16 kDa found in eukaryotic species. Objective: The aim of this study was to generate monoclonal antibody against Cro s 2 in order to char-acterize this major allergen of saffron pollen. Methods: BALB/c mice were immunized to obtain adequate humoral response. Splenocytes were prepared from the immunized animals, mixed with the P3-X63-Ag8.653 myeloma cells and fused by means of PEG 1500. After two weeks of culturing in HAT-containing media, the supernatant from those wells growing hybridomas were screened by ELISA using plates coated with Cro s 2. Cells from positive wells were cloned at least 3 times by limiting dilution. Specific-ity and cross-reactivity of the mAbs were determined by Western blot analysis and sandwich ELISA. Results: Two stable hybridoma clones secreting mAbs against Cro s 2 were obtained and expanded. The anti-Cro s 2 mAbs were also found to cross-react with other plant profilins. Isotype of this mAb was identified as μ heavy chain and k light chain. Conclusion: The anti-Cro s 2 mAb could be a useful tool for characteriza-tion and standardization of many pollen and fruit-derived profilins.
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 ...
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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 ...
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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.
Ezzatallah Basiri; Sadreddin Mohseni Ardehali; Mehrnoosh Doroudchi; Fereidoun Mahboodi; Arsalan Kharazmi; Abbas Ghaderi
Volume 1, Issue 3 , December 2004, , Pages 194-199
Abstract
Background : Production of monoclonal antibodies to Leishmania antigens assists the identification and characterization of these organisms. Objective: Production of monoclonal antibodies against epitopes on the gp63. Methods: Two murine monoclonal antibodies to gp63 were produced and characterized. The ...
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Background : Production of monoclonal antibodies to Leishmania antigens assists the identification and characterization of these organisms. Objective: Production of monoclonal antibodies against epitopes on the gp63. Methods: Two murine monoclonal antibodies to gp63 were produced and characterized. The reactions of both antibodies with soluble leishmanial antigens, purified gp63 and truncated recombinant gp63 molecules were studied by an ELISA assay. These two antibodies reacted with the crude soluble antigens prepared from 4 reference strains of Leishmania, 10 isolates from the patients, purified gp63 and recombinant gp63 molecules. However, no reaction with several non-leishmanial antigens was observed. Reaction of both antibodies with the intact recombinant gp63 and truncated molecules were compared. Results: The results indicated that the two antibodies specifically recognize two different epitopes on the gp63 molecule. Conclusion: Possible applications of such antibodies in searching for immunogenic epitopes are discussed.