Distinct antibody was eluted from the column through washing buffer containing 50 mM Nacl, and the fractions were collected in 5 ml/20 ml

Distinct antibody was eluted from the column through washing buffer containing 50 mM Nacl, and the fractions were collected in 5 ml/20 ml. 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 Befiradol of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases. Keywords:Monoclonal antibody, Human IgG, Balb/c mice, ELISA == Introduction == Monoclonal antibodies can be produced in specialized cells through a method now popularly known as hybridoma technology.1Hybridoma technology was first invented by two scientists, Georges Kohler and Befiradol Cesar Milstein. Befiradol From 1975, Khler and Milstein successfully fused antibody- producing mouse spleen cells with mouse myeloma cells, the fusion of somatic cells has been carried out for many years with a variety of different aims and this technique quickly became one of immunologys key technologies.2Using hybridoma technology, monoclonal antibodies have been prepared against a wide range of antigens including growth factors, growth factor receptors, viruses, bacterial products, hormones and differentiated antigens.3 In fact, monoclonal antibodies (mAbs) have been widely applied in various fields such as diagnosis applications, purification, disease monitoring, identifying prognostic markers and therapy.4For most research, diagnostic and therapeutic purposes, monoclonal antibodies derived from a single clone and thus specific for a single epitope, are preferable.5 In most diagnostic kits of the infectious diseases, for instance, Rubella, H.pylor Toxoplasmosis and etc, the conjugated monoclonal antibodies against human IgG carry out as a key role. Due to in this need, generation of mAb seems invaluable. To approach these goals, generation and characterization of a highly specific mAb against human IgG was investigated. The prim aim of this study including: production and application of mabs against human IgG for development of diagnostic kits and large scale, semi-industrial production and standardization of this product towards self-sufficiency of the country. == Materials and Methods == == Immunization Procedure and Screening of Immunized Animals == Four female Balb/c (6-8 weeks old) mice were used for purified human IgG (Affinity Purified Human IgG, Sigma) immunization. Each mouse was immunized 4 times with an interval of two-three weeks subcutaneously. The first Immunization was performed using Freund’s complete adjuvant (Sigma-Aldrich Co. Louis, USA). Incomplete Freund’s adjuvant (Sigma-Aldrich Co. Louis, USA) was used for the 2nd, 3rdand 4thimmunization. For the first immunization, 50 g of purified human IgG was mixed with an equal volume of Freund’s complete adjuvant. For the subsequent immunizations 50 g purified human IgG were injected with Freund’s incomplete adjuvant. Three days before the cell Befiradol fusion, 50 g of Mouse monoclonal to VCAM1 antigen (without any adjuvant) was injected intravenously. A week after the second injection, blood was taken from each mouse by a vertical incision of the tail vein and the antibody response was measured by ELISA. The mouse with the highest serum antibody titre was selected as the spleen donor. Sera collected from non-immunized and immunized mice served as negative and positive controls. == Cell Fusion and Hybridoma Production == Three days Befiradol after final immunization, spleen of the immunized mouse was aseptically removed and fused with SP2/0 myeloma cell line at a ratio of 1 1:5 (1 SP2/0 and 5 spleen cells) by PEG (polyethyleneglycol, MW 1450, Sigma) as fusogen. Selective HAT medium (Gibco) was added to the fused cells and cells were seeded into five 96-well microtitre plates (Nunc) containing feeder layer. The cells were incubated at 37 C with 5% CO2 for 2-3 days. Cell growth and colony formations were examined daily. Colonies were appeared after 5-7 days. Once the colony diameter reached to 1 1 mm the presence of antibody against the immunized antigen was determined by ELISA method. Then, positive hybridomas were selected for.