%0 Journal Article
%T Optimization of Unnicked 汕2-Glycoprotein I and High Avidity Anti-汕2-Glycoprotein I Antibodies Isolation
%A Andrej Artenjak
%A Adrijana Leonardi
%A Igor Kriˋaj
%A Aleˋ Ambroˋiˋ
%A Snezna Sodin-Semrl
%A Borut Boˋiˋ
%A Saˋa ˋuˋnik
%J Journal of Immunology Research
%D 2014
%R 10.1155/2014/195687
%X Patient biological material for isolation of 汕2-glycoprotein I (汕2GPI) and high avidity IgG anti-汕2-glycoprotein I antibodies (HAv anti-汕2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked 汕2GPI and HAv a汕2GPI to gain unmodified proteins in higher yields/purity. Isolation of 汕2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv a汕2GPI affinity chromatographies with immobilized protein G and human 汕2GPI 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 汕2GPI increased from 6.56ˋmg to 9.94ˋmg. In the optimized isolation procedure the high molecular weight protein (proteoglycan 4) was successfully separated from 汕2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonal HAv anti-汕2GPI from different matrixes was 13.8%, as determined by our in-house anti-汕2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked 汕2GPI and HAv anti-汕2GPI, improving the purity of antigen and antibodies as well as increasing the number of tests routinely performed with the in-house ELISA by ~50%. 1. Introduction Recent findings of antiphospholipid syndrome (APS) pathogenesis support the important role of 汕2-glycoprotein I (汕2GPI), as one of the most studied antigens [1每3]. 汕2GPI is a ~50ˋkDa protein with a mean plasma concentration in the healthy population of ~180ˋmg/L. The protein consists of 326 amino acids folded into 5 domains [4, 5]. The first 4 domains contain approximately 60 amino acids, whereas the last, 5th domain consists of 82 amino acids containing specific segments of positively charged amino acids 281CKNKEKKC288 and a hydrophobic loop 313LAFW316, which along with 19 amino acids of the C-terminal extension form the binding site to negatively charged phospholipids [6, 7]. Plasmin can clip/nick 汕2GPI at amino acids L317T318 and consequently terminate its ability to bind phospholipids [8]. Furthermore, recently it was observed that 汕2GPI can exist in different conformations, that is, in a circular form that can change to an open (fishhook) conformation after exposure to anionic structures or negatively charged phospholipids, which can be stabilized by anti-汕2GPI antibodies (anti-汕2GPI) [9, 10]. The presence of anti-汕2GPI in human
%U http://www.hindawi.com/journals/jir/2014/195687/