title: Production and Purification of a Polyclonal Antibody Against Purified Mouse IgG2b in Rabbits Towards Designing Mouse Monoclonal Isotyping Kits.
abstract: OBJECTIVE
  Mouse IgG subclasses containing IgG1, IgG2a, IgG2b and IgG3 have been defined and described both physiochemically and immunologically.
  
  
  METHODS
  Sepharose beads conjugated with protein A affinity chromatography was used for purification of mouse IgG2b. Sodium citrate buffer (0.1 M, pH: 3.5) was used for separation of mouse IgG2b. Verification of the purified fractions was monitored by SDS-PAGE (polyacrylamide gel electrophoresis) in reducing condition. Immunized rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. After dialysis against tris-phosphate buffer (pH: 8.1) ion exchange chromatography column was used for purification of rabbit anti-mouse IgG2b. The periodate method was performed for conjugation with some variations. After conjugation, direct ELISA was used to determine the titer of HRP conjugated rabbit IgG against mouse IgG2b.
  
  
  RESULTS
  The titer of rabbit anti-mouse IgG2b that determined by ELISA was 32000. The purity of rabbit anti-mouse IgG2b was about 95%. The optimum dilution of prepared HRP conjugated IgG was 1:10000. This study showed that ion-exchange chromatography and affinity chromatography could be appropriate techniques for purification of mouse IgG and IgG subclasses respectively.
  
  
  CONCLUSIONS
  This study showed that affinity chromatography could be an appropriate method for purification of IgG2b antibodies.
authors: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors: Sadeq
authors: S
authors: Eivazi
authors: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
authors: Jafar
authors: J
authors: Majidi
authors: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
authors: Leili
authors: L
authors: Aghebati Maleki
authors: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors: Jalal
authors: J
authors: Abdolalizadeh
authors: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
authors: Mehdi
authors: M
authors: Yousefi
authors: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors: Majid
authors: M
authors: Ahmadi
authors: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors: Somayeh
authors: S
authors: Dadashi
authors: Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors: Zahra
authors: Z
authors: Moradi
authors: Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
authors: Elmira
authors: E
authors: Zolali
keywords: Affinity chromatography; Horse radish peroxidase conjugation; Ion-exchange chromatography; Mouse IgG2b