Preparation And Evaluation Of Rabbit Anti-Buffalo Immunoglobulin Antibody Peroxidase Conjugate
By: Muhammad Babar
| Prof.Dr.Muhammad Ashraf.
Contributor(s): Mr. Shahid Abbas | Prof. Dr | Faculty of Biosciences.
Material type: 
BookPublisher: 2005Subject(s): Department of PhysiologyDDC classification: 0926,T Dissertation note: Enzyme linked immunosorbent assay (ELISA) is one of the most sensitive rapid and reliable techniques for diagnosis of infectious diseases. For execution of ELISA, antibody-peroxidase conjugate is the fundamental reagent. Turnip peroxidase was purified from turnips, that includes homogenization, inactivation of catalase, ammonium sulphate precipitation and size exclusion chromatography on Sephadex G-25-80. The purified peroxidase had Rz value of 1.7, total protein 0.9 mg/ml and total enzyme activity 36152 units/liter. The buffalo serum Ig-G was fractionated using 40 percent final concentration of ammonium sulphate followed by anion exchange chromatography. The salt fractionated serum globulins (10 ml) was depleted of its Ig-G in less than 25 minutes on DEAE cellulose packed column followed by suitable elution. The Ig-G solution (1.0 gm/dl) was mixed in four times volume of oil base (Liquid paraffin and emulsifiers). Rabbits were primed and boosted (0.25ml/: subcut) with buffalo Ig-G antigen with 21 days interval. The immune serum was harvested on 21 days post-boosting. The serum contained 2048 agar gel precipitation AGP units and 10,000 ELISA units. Rabbit anti buffalo Ig-G was purified with salt precipitation followed by anion exchange chromatography. The peroxidase was linked with the rabbit anti-buffalo Ig-G using the sodium metaperiodate. The conjugate was titrated against buffalo Ig-G and working dilution for execution of ELISA was 1: 2000.
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Thesis
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UVAS Library Thesis Section | Veterinary Science | 0926,T (Browse shelf) | Available | 0926,T |
Enzyme linked immunosorbent assay (ELISA) is one of the most sensitive rapid and reliable techniques for diagnosis of infectious diseases. For execution of ELISA, antibody-peroxidase conjugate is the fundamental reagent. Turnip peroxidase was purified from turnips, that includes homogenization, inactivation of catalase, ammonium sulphate precipitation and size exclusion chromatography on Sephadex G-25-80. The purified peroxidase had Rz value of 1.7, total protein 0.9 mg/ml and total enzyme activity 36152 units/liter. The buffalo serum Ig-G was fractionated using 40 percent final concentration of ammonium sulphate followed by anion exchange chromatography. The salt fractionated serum globulins (10 ml) was depleted of its Ig-G in less than 25 minutes on DEAE cellulose packed column followed by suitable elution. The Ig-G solution (1.0 gm/dl) was mixed in four times volume of oil base (Liquid paraffin and emulsifiers). Rabbits were primed and boosted (0.25ml/: subcut) with buffalo Ig-G antigen with 21 days interval. The immune serum was harvested on 21 days post-boosting. The serum contained 2048 agar gel precipitation AGP units and 10,000 ELISA units. Rabbit anti buffalo Ig-G was purified with salt precipitation followed by anion exchange chromatography. The peroxidase was linked with the rabbit anti-buffalo Ig-G using the sodium metaperiodate. The conjugate was titrated against buffalo Ig-G and working dilution for execution of ELISA was 1: 2000.
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