Purification of anti-chymotrypsin antibodies for the preparation of a bioaffinity matrix with oriented chymotrypsin as immobilized ligand |
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| Institution: | 1. Department of Biological and Biochemical Sciences, Department of Analytical Chemistry, University of Pardubice, Náměsti Čs. legií 565, 532 10 Paradubice, Czech Republic;2. Department of Pathological Physiology, 1st Faculty of Medicine, Charles University, U nemocnice 5, 128 53 Prague 2, Czech Republic;3. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic;1. CNR-IMM, Via S. Sofia 64, 95123 Catania, Italy;2. CNR-IPCB, Via Paolo Gaifami 18, 95126 Catania, Italy;3. CNR-IPCB, Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy;1. Shandong Provincial Key Laboratory of Molecular Engineering, School of Chemistry and Chemical Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, PR China;2. Shandong University of Political Science and Law, Jinan 250014, PR China;3. Gubkin University, Department of Physical and Colloid Chemistry, 65, Leninsky Prospekt, Building 1, Moscow 119991, Russian Federation;4. Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China;1. Hacettepe Universty, Faculty of Science, Department of Chemistry, Ankara, Turkey;2. Hacettepe University, Institute of Science, Nanotechnology and Nanomedicine Division, Ankara, Turkey;1. College of Resources and Environment, The Key Laboratory of Straw Biology and Utilization, The Ministry of Education, Jilin Agricultural University, Changchun, 130118, China;2. Institute of Mathematica, Jilin Normal University, Siping, Jilin, 136000, China;3. College of Chemistry and Chemical Engineering, Chifeng University, Chifeng, 024000, China |
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| Abstract: | Polyclonal antibodies suitable for the oriented immobilization of chymotrypsin we prepared by chromatography on a bioaffinity matrix which had the enzyme immobilized through its active site to antilysin, covalently linked to bead cellulose. After periodate oxidation of their carbohydrate moieties, the isolated antibodies were coupled to a hydrazide derivative of bead cellulose. The periodate oxidation step, which led to greater efficiency and stability of the immunosorbent, had no deleterious effect on antibody activity as assessed by ELISA. Addition of chymotrypsin to the immunosorbent yielded an enzymically active bioaffinity matrix with the optimum molar enzyme/antibody ratio of 2. |
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