Patient biological material for isolation of β2-glycoprotein I (β2GPI) and high avidity IgG anti-β2-glycoprotein I antibodies (HAv anti-β2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked β2GPI and HAv aβ2GPI to gain unmodified proteins in higher yields/purity. Isolation of β2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv aβ2GPI affinity chromatographies with immobilized protein G and human β2GPI 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 β2GPI increased from 6.56？mg to 9.94？mg. In the optimized isolation procedure the high molecular weight protein (proteoglycan 4) was successfully separated from β2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonal HAv anti-β2GPI from different matrixes was 13.8%, as determined by our in-house anti-β2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked β2GPI and HAv anti-β2GPI, 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 β2-glycoprotein I (β2GPI), as one of the most studied antigens [1–3]. β2GPI is a ~50？kDa protein with a mean plasma concentration in the healthy population of ~180？mg/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 β2GPI at amino acids L317T318 and consequently terminate its ability to bind phospholipids [8]. Furthermore, recently it was observed that β2GPI 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-β2GPI antibodies (anti-β2GPI) [9, 10]. The presence of anti-β2GPI in human
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