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ANALYTICAL TRICKS AND TIPS
THE BIOLOGICAL CONTEXT
Specificity, sensitivity etc.
PROs and CONTROs
Tesi Mancini 2006
CDT in sepsis
Int J Hematol. 2010 Mar;91(2):238-44. Epub 2010 Feb 5.
Glycosylation-modified erythropoietin with improved half-life and biological activity. 2010
Su D, Zhao H, Xia H.
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No 3 A1 Road 10, Shenyang Economy and Technology Development Zone, Shenyang 110027, China. email@example.com
Erythropoietin (EPO) controls the production of red blood cells, so it is important to maintain high levels of EPO activity and half-life. Here, we modified glycosylation sites in human erythropoietin (HuEPO) gene, resulting in proteins with addition of 1-4 glycosylation sites. The modified gene was introduced into CHO cells. The expressed EPO analogs were analyzed by SDS-PAGE. Half-life of the analogs was determined by sialic acid content test. In vivo potencies of analogs were evaluated by reticulocyte count and haematocrit level. The metabolic clearance of recombinant human erythropoietin (rHuEPO) and its analogs were determined by EPO immunoradiometrics assay. We have shown that the carbohydrate content in modified EPO molecules is increased. The modified EPO, [Val(3)Asn(4)Thr(6)Asn(30)Thr(32)Val(87)Asn(88)Thr(90)]EPO, increases 3.3 times in elimination half-life, 2.1 times in activity and prolongs 2 days functional time in vivo in comparison to rHuEPO. These findings suggest that the addition of glycosylation sites in EPO enhances half-life and biological activity of EPO, duration of action of EPO anlogues positively correlated with the number of glycosylated sites, while addition of 4 glycosylation sites does not further enhance the erythropoietic potency.