The B-cell CD20 antigen is one of the most reliable surface targets in immunotherapy of B lymphoma. In this project, we studied the production and characterization of a new monoclonal antibody against chimeric human CD20 extra loops (hCD20 exl). The results showed that clone C12H, IgG2/k isotype reacted with the antigen in ELISA and immunoblot. The Kd value was found to be 2´10-9M and flow cytometry results showed that 99.9% and 99.7% of the Daudi and Raji cells respectively were stained with C12H monoclonal antibody (mab) but not with Jurkat cell lines. It also effectively competed with Rituximab, thus, the staining of the Daudi and Raji cell lines was reduced to 55.9% and 40.5% of cells respectively. Based on the high affinity reaction of C12H mab and appropriate reactivity of C12H mab with the native antigen on the surface of Raji and Daudi cells in flow cytometry, it was concluded that development and evaluation of C12H mab could be a beneficial candidate for further application in genetically engineered monoclonal antibody.

Background & Aims: 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 specific binding of these antibodies was determined by immunological assay such as ELISA and Immunofluorescence and flowcytometry.Results: After cell fusion and screening, one desired monoclone achieved with absorbance about 2. Protein-A-Sepharose column affinity chromatography yielded about 5 mg of purified monoclonal antibody. The SDS-PAGE results confirmed purification of antibody. The result of ELISA, direct immunofluorescence staining and flow cytometry confirmed specific attachment to CD20.Conclusions: These results indicate that such monoclonal antibodies against CD20 can be used in diagnosis of CD20 in the cells surface by immunological assay such as ELISA and Immunofluorescence and flowcytometry.

Nowadays, anti-CD20 antibodies are being used in the therapy of lymphomas in free or radiolabeled form. Conjugation process was performed using native antibody and DOTA-NHS compound, synthesized in our laboratory. Thin layer chromatography and gel filtration were used to control the reaction progress as well as purification of the final antibody conjugate. 67GaCl3 produced at AMIRS was used to label the immunoconjugate and the reaction conditions were optimized for time, temperature and reactant concentrations. Quality control of the radioimmunoconjugate was performed using RTLC and gel filtration. The radiochemical purity was shown to be over 95%. The radioimmunoconjugate was used in the biodistribution studies up to 28 h and it was shown that after 24h most of the activity was accumulated in reticuloendothelial system consisting liver and spleen.

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.

Background: CD20 is an important cell surface receptor that is used for target therapy of B cell lymphoma and some related blood diseases due to vital function of CD20.In previous studies, a Rituximab based humanized single chain variable fragment (scFv) antibody showed good reactivity against B cell related cancer cells. But this recombinant protein produced Inclusion Bodies (IBs) inEscherichia coli (E. coli) cytoplasm.The aim of this study was to investigate the effect of coexpression with cytoplasmic chaperones on expression and solubility of humanized anti-CD20 scFv inE.coli.Methods: For this purpose, the fragment coding for anti-CD20 huscFv subcloned into the pET22b (+) and transformed into theE. coli BL21 (DE3) was evaluated. In order to inhibit the production of IBs, the effects of co-expression with cytoplasmic chaperones GroEL, DnaK, GroES, Tig, DnaJ and GrpE were investigated.Result: Coexpression with cytoplasmic chaperones led to increased soluble expression of anti-CD20 recombinant protein. Among investigated chaperones, pKJE7 chaperone plasmid containing DnaJ, GrpE, DnaK chaperone genes had significant effects with an expression yield of 325μg/ml soluble anti-CD20 scFv.Conclusion: The result of this study demonstrated remarkable effect of pKJE7 chaperone on enhancement of soluble expression of anti-CD20 huscFv antibody inE. coli.

Immunodeficiency and autoimmune disease may occur concomitantly in the same individual. Some of the immunodeficiency syndromes, especially humoral defects are associated with autoimmune disorders. Hematological manifestations such as thrombocytopenia and hemolytic anemia are the most common presentations. Persistent antigen stimulation due to an inherent defect in the ability of the immune system to eradicate pathogens is the primary cause leading to autoimmunity in patients with primary immunodeficiency states.We describe a 10 year old Iranian girl with chronic granulomatous disease -the autosomal recessive type with mutation of NCF1 gene P47- associated with selective IgA deficiency, refractory immune thrombocytopenia that showed an excellent response to Rituximab (Anti- CD20 monoclonal antibody).Patients with primary immunodeficiencies may have variable autoimmune manifestations. So for early detection and appropriate treatment, autoimmune diseases should always be suspected in such patients.

Neuromyelitis optica (NMO) is an autoimmune inflammatory disease of the central nervous system with preferential involvement in the optic nerve and spinal cord with a widespread spectrum of clinical features; multiple therapeutic agents have been used with different results. Recent evidence points to B‑cell‑mediated humoral immunity in the pathogenesis of NMO. Rituximab targets the CD20 antigen on B‑cells. Treatment leads to profound B‑cell depletion, principally over an antibody‑dependent cell cytotoxicity mechanism. The aim of our study was to review clinical trials to elucidate the impact of rituximab on the relapse rate, Expanded Disability Status Scale (EDSS), and progression of disability in NMO. We performed a comprehensive review of all studies that evaluated clinical and paraclinical effects of rituximab on NMO. MEDLINE‑PubMed, Web of Sciences, EMBASE, and Cochrane databases up to June 2016 included in our searches. In addition, reference lists from articles identified by search as well as a key review article to identify additional articles included in the study. Rituximab targets the CD20 antigen on B‑cells and decreases attack frequency and severity in patients with NMO; however, it does not remove attacks, even when modifying treatment to achieve B‑cell depletion. Most of the investigations revealed that EDSS significantly in all patients with rituximab treatment will be decreased after treatment with rituximab. No new or enlarged lesions or pathological gadolinium enhancement was observed in serial brain and spinal cord magnetic resonance imaging, except for those observed concomitantly with clinical relapses and the median length of spinal cord lesions was significantly reduced after therapy. Rituximab targets the CD20 antigen and decreases attack frequency and severity in patients with NMO.