Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation

Patient biological material for isolation of beta 2-glycoprotein I (beta 2GPI) and high avidity IgG anti-beta 2-glycoprotein I antibodies (HAv anti-beta 2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked beta 2GPI and HAv a beta 2GPI to gain unmodified proteins in higher yields/purity. Isolation of beta 2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv a beta 2GPI affinity chromatographies with immobilized protein G and human beta 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 beta 2GPI 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 beta 2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonal HAv anti-beta 2GPI from different matrixes was 13.8%, as determined by our in-house anti-beta 2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked beta 2GPI and HAv anti-beta 2GPI, improving the purity of antigen and antibodies as well as increasing the number of tests routinely performed with the in-house ELISA by similar to 50%.