Preparation of Fab Fragment Immobilized Immunoaffinity Cryogels and Albumın Purification
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2017-01-19Yazar
Hüseynli, Sabina
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Albumin, composed of a single chain of 585 amino acids, is synthesized in the liver. It is the most common protein found in blood plasma of humans and other mammals. It constitutes 60% of the proteins found in blood. It is also found in tissue fluids, especially in muscle and in the skin, small amounts in tears, sweat, stomach juices and in the gall. 30-40% of the total albumin in the body is in the blood. The most important function is to balance the water between the blood and the tissue fluids, as well as to keep the fat acids and various other substances moving. Albumin is carrier protein that organizes oncotonic pressures in the body and provides inter-tissue substance transport.
In affinity chromatography, the interaction of the corresponding antibodies with antigens, enzymes with substrate anologues and receptors of hormones is the type of interaction. Immunoaffinity chromatography, a type of affinity chromatography, is a chromatographic technique frequently used in recent years. Antibodies used as ligands in immunoaffinity chromatography are used for the purification of antigens.
In this work, cryogel columns immobilized Fab fragments were prepared for human serum albumin (HSA) purification. For this purpose, firstly enzymatic hydrolysis of anti human serum albumin (Anti-HSA) molecules with papain enzyme was performed and the resulting Fab fragments were immobilized in the cryogel column. For control purposes, Fab fragment immobilized PHEMAC cryogel column was prepared. Characterization studies of prepared columns (SEM, BET, FTIR-ATR swelling) were performed. The effect of various parameters (pH scanning, concentration, flow rate, etc.) on the albumin purification process was examined.
Albumin selectivity depends on changes in the Fab region. Thus, instead of immobilizing the whole of the molecule, the functional Fab region responsible for antigen binding is immobilized. Thus, both the immobilization process is facilitated and the efficiency of the column is increased. PHEMA-based cryogels are biocompatible with proteins. In this study, HSA purification was carried out selectively from aqueous solution.