Author/Editor		Artenjak, Andrej; Leonardi, Adrijana; Križaj, Igor; Ambrožič, Aleš; Sodin-Šemrl, Snežna; Božič, Borut; Čučnik, Saša
Title		Optimization of unnicked ß2-glycoprotein I and high avidity anti-ß2-glycoprotein I antibodies isolation
Type		članek
Publication year		2014
Language		eng
Abstract		Patient biological material for isolation of ß2-glycoprotein I (ß2GPI) and high avidity IgG anti-ß2-glycoprotein I antibodies (HAv anti-ß2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked ß2GPI and HAv aß2GPI to gain unmodified proteins in higher yields/purity. Isolation of ß2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv aß2GPI affinity chromatographies with immobilized protein G and human ß2GPI were used. The unknown protein found during isolation was identified by liquid chromatography electrospray ionization mass spectrometry and the nonredundant National Center for Biotechnology Information database. The average mass of the isolated unnicked purified ß2GPI increased from 6.56mg to 9.94mg. In the optimized isolation procedure the high molecular weight protein (proteoglycan 4) was successfully separated from ß2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonalHAv anti-ß2GPI from differentmatrixes was 13.8%, as determined by our in-house anti-ß2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked ß2GPI and HAv anti-ß2GPI, improving the purity of antigen and antibodies as well as increasing the number of tests routinely performed with the in-house ELISA by 50%.
Keywords		beta2 glikoproteini izolacija protiteles izolacija proteina G ELISA