Sadeq Eivazi1, Jafar Majidi2, Leili Aghebati Maleki2, Jalal Abdolalizadeh3, Mehdi Yousefi2, Majid Ahmadi1, Somayeh Dadashi1, Zahra Moradi1, Elmira Zolali4. 1. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 2. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. 3. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 4. Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract
PURPOSE: 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. CONCLUSION: This study showed that affinity chromatography could be an appropriate method for purification of IgG2b antibodies.
PURPOSE: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. CONCLUSION: This study showed that affinity chromatography could be an appropriate method for purification of IgG2b antibodies.