Title of article :
Polyclonal Antibody Production against Mouse Purified IgG2a towards Use in Basic Research
Author/Authors :
Moradi nebrin ، Zahra Immunology Research Center, Faculty of Medicine - Tabriz University of Medical Sciences , Majidi ، Jafar Department of Immunology - Immunology Research Center, Faculty of Medicine - Tabriz University of Medical Sciences , Aghebati Maleki ، Leili Department of Immunology - Immunology Research Center, Faculty of Medicine - Tabriz University of Medical Sciences , Kazemi ، Tohid Department of Immunology - Immunology Research Center, Faculty of Medicine - Tabriz University of Medical Sciences , Abdolalizadeh ، Jalal Department of Immunology, Immunology Laboratory - Faculty of Medicine, Drug Applied Research Center - Tabriz University of Medical Sciences , Dadashi ، Somaieh Department of Immunology - Immunology Research Center, Faculty of Medicine - Tabriz University of Medical Sciences , Eyvazi ، Sadeg Department of Immunology - Immunology Research Center, Faculty of Medicine - Tabriz University of Medical Sciences , Ahmadi ، Majid Department of Immunology - Immunology Research Center, Faculty of Medicine - Tabriz University of Medical Sciences , Majidi zolbanin ، Naiemeh Pharmacology Department - Faculty of pharmacy - Tabriz University of Medical Sciences
Abstract :
Background: The ability of polyclonal antibodies to react with many epitopes of an antigen makes them valuable reagents in research and diagnosis. The aim of this study was purification of mouse IgG2a and production of polyclonal antibody against purified mouse IgG2a subclass. Materials and Methods: Mouse IgG2a was purified by ProA affinity. Verification method of the purified antibody was SDSPAGE and ELISA by a mouse isotyping Kit. Rabbit was immunized with purified IgG2a. The production of antibody in rabbit was investigated by direct ELISA method. Rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. Polyclonal antibody was purified by ionexchange chromatography and labeled with HRP. The titre and cross reactivity of product was detected by direct ELISA method. Results: The results of SDSPAGE in reduced and nonreduced conditions showed bands with 50KDa, 2530 KDa MW and a distinct band with 150 KDa MW. Isotype determination showed the presence of mouse IgG2a in related fraction. The titer of Antimouse polyclonal antibody was 200000. The optimum titer of prepared HRP conjugated IgG was 4000. Conjugated rabbit IgG has more cross reactivity with mouse IgG2b. Conclusion: Taking together, affinity chromatography and ionexchange chromatography are appropriate techniques for purification of mouse IgG subclasses and rabbit IgG, respectively.
Keywords :
Affinity chromatography, purification, polyclonal antibody, IgG2a, ProA , ProG
Journal title :
Research in Molecular Medicine
Journal title :
Research in Molecular Medicine
