Introduction: Leishmania (L. ) infantum is the etiologic cause of visceral leishmaniasis in Iran. Efficient vaccines and diagnosis methods are required to control leishmaniasis. The aim of this study is produce and optimize MONOCLONAL antibodies against promastigotes forms of L. infantum antigen. Materials and Methods: The mice were vaccinated with the L. infantum antigen and their ANTIBODY titers were determined by the ELISA method. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol. The effect of supernatant of SP2/0 and mice peritoneum macrophage cells culture (SSMCC) on hybridoma cell proliferation was studied. Results: Among the 12 fusion, a total of 26 MONOCLONAL were positive. 12 of which had acceptable optical absorbance in OD 450 nm. Finally, 4 clones, designated as 8D2 FVI6, 8D2 FVI3, 6G2 FV4 and 6G2 FV3. From these hybrids, antipromastigotes L. infantum MONOCLONAL antibodies were obtained. SSMCC was shown to play a key role in hybridoma proliferation and of mAb production. It seemed that SSMCC is rich of growth factors. Conclusion: It seems in the near future, this SCCSM can be used as a growth factor for cancerous and non-cancerous cells in research centers at a wider level.

Introduction: MONOCLONAL ANTIBODY has many applications against peroxidase among which peroxidase-anti peroxidase (PAP) complex formation is of great importance. This complex is used in many immunohistochemical and immunocytochemical staining techniques. The aim of this study was the production of a mouse antiperoxidase of hybridoma cell line to be used in peroxidase - anti peroxidase immunostaining.Materials & Methods: The Balblc mice were first immunized by repeated injections of horseradish peroxidase (HRP). After the confirmation of their immunization by ELISA, the spleen lymphocytes and SP2/0 myeloma cells were hybridized by using PEG. The hybridoma cells were then selected through the selective HAT medium. From seven rounds of cell fusion, 224 hybridoma clones were obtained, from which six clones produced MONOCLONAL ANTIBODY against HRP. Once again from these six clones, two clones were selected and developed. Limiting dilution was performed twice and monospecific clones were developed.Results: Using the Isostrip kit revealed that both MONOCLONAL anti peroxidase antibodies (P1F11D2, P2F6F3) were from lgG1 isotype class. In addition, ELISA test showed that the MONOCLONAL antibodies don't affect the active site of the enzyme. Therefore it seems that the produced antibodies are suitable for formation of PAP immune complex.Conclusion: At present, this study is being carried out farther to produce PAP complex and investigate its practical usage in immunohistochemical and immunocyto chemical techniques.

Immunoglobulin E (IgE) is one of the five classes of Immunoglobulin. MONOCLONAL Antibodies (mabs) have been produced by a single clonotype to different epitops of IgE. The potential application of mab is widly extended including diagnostic kits. In this study, we attempt to produce mab against Human IgE. Balblc mice were immunized with purified IgE and spleen cells fused with SP2/0 mouse myeloma cell line in the presence of poly ethylen glycol (PEG). Supernatant of hybridoma cells screened with Enzym Linked Immonosorbent Assay (ELISA) Method. Cloning of selective high absorbanc wells achieved with Limiting Dilution (L.D) Method. The best Clone (Monoclone) was selected by Elisa and approved by Immunoblot. The subclass of the choice mab was determined and the clone freezed in -196 degree liqid nitrogen. The results showed that final production of three successful fusions were 156 positive wells with high absorbance in reaction with IgE. Fourteen wells with the highest absorbance selected for cloning. Over the 100 well, which produced mabs the G10F7 was the best one which, displayed the high absorbance in reaction with IgE and the lowest cross-reactivity (about negative control) against IgM, IgG and IgA. These results were approved by presence of high density bond in reaction with IgE in Immunobloting. This mab was shown to be IgG1 subclass with kappa light chain.      

Introduction: Production of MONOCLONAL ANTIBODY against Alkaline phosphatase for application in immunohistochemical and immunocytochemical techniques such as alkaline phosphatase anti-alkaline phosphatase (APAAP) method.   Material and Methods: In this investigation female Balb/c mice were immunized by several injections of alkaline phosphatase and the ANTIBODY titer in their sera were measured after each injection. The spleen lymphocytes of immunized mice and Sp2/0 myeloma cells were fused using 50% polyethylen glycol as fusing agent and hybridoma cells were selected by HAT medium. Identification and selection of anti-alkaline phosphatase producing clones were done by performing ELISA test on supernatants of all the resulting clones. Limiting dilution was performed twice on antiboby producing clones for their seperation and the resulted subclones were propagated. Since APAAP complex must be enzymatically active for using in immunohistochemical techniques the adhesion of Ab molecule to enzyme molecule must not affect the enzyme activity. For investigation of this effect, an ELISA technique was planned and the supernatants of selected hybridoma clones were studied by this method. For production of concentrated Ab the hybridoma cells were injected to peritoneal cavity of mice and the ascetic fluids were obtained. Finally the antibodies isotypes were determined.   Results: After 6 fusion experiments 104 hybridoma clones were abtained and two clones (A1G9 and A1G8) which were ANTIBODY producing and had the highest absorbance in ELISA test were selected. Using the limiting dilution method finally two MONOCLONAL subclones A1G8F7 and A1G9G3 were selected. ELISA experiments showed that antibodies which were produced by selected hybridoma clones did not react with active site of the enzyme and did not interfer with enzymatic activity. Electrophoresis of ascetic fluids of hybridoma injected mice showed a prominent band in γ position. Isotype determination of MONOCLONAL antibodies showed that both hybridoma clones produce ANTIBODY from IgG class with k light chain.   Conclusion: Because MONOCLONAL antibodies which are produced by the obtained hybridoma cell lines are from IgG class and do not affect the enzyme activity, it seem's that they are suitable for APAAP complex formation. Other steps of this study are now being performed until APAAP complex formation and it's application in immunohistochemistry.  

We have employed a peptide-based ANTIBODY generation protocol for producing ANTIBODY against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glycosylation sequences, we generated a mouse MONOCLONAL ANTIBODY capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this ANTIBODY. This ANTIBODY is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays.