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

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]  Aaas News,et al.  Book Reviews , 1893, Buffalo Medical and Surgical Journal.

[2]  S. Čučnik,et al.  High Avidity Anti-β2-Glycoprotein i Antibodies Activate Human Coronary Artery Endothelial Cells and Trigger Peripheral Blood Mononuclear Cell Migration , 2013 .

[3]  T. Schmidt,et al.  Molecular weight characterization of PRG4 proteins using multi-angle laser light scattering (MALLS). , 2013, Osteoarthritis and cartilage.

[4]  T. Schmidt,et al.  Diminished cartilage-lubricating ability of human osteoarthritic synovial fluid deficient in proteoglycan 4: Restoration through proteoglycan 4 supplementation. , 2012, Arthritis and rheumatism.

[5]  Y. Shoenfeld,et al.  Antiphospholipid antibodies as non-traditional risk factors in atherosclerosis based cardiovascular diseases without overt autoimmunity. A critical updated review. , 2012, Autoimmunity reviews.

[6]  S. Čučnik,et al.  Avidity of anti-β2-glycoprotein I antibodies in patients with antiphospholipid syndrome , 2012, Lupus.

[7]  T. Kveder,et al.  Autoimmune reactivity of IgM acquired after oxidation , 2011, Redox report : communications in free radical research.

[8]  Urška Žager,et al.  Immunochemical properties and pathological relevance of anti-β₂-glycoprotein I antibodies of different avidity. , 2011, International immunology.

[9]  S. Čučnik,et al.  The avidity of anti-β2-glycoprotein I antibodies in patients with or without antiphospholipid syndrome: a collaborative study in the frame of the European forum on antiphospholipid antibodies , 2011, Lupus.

[10]  P. Meroni,et al.  Pathogenesis of antiphospholipid syndrome: understanding the antibodies , 2011, Nature Reviews Rheumatology.

[11]  P. D. de Groot,et al.  Beta2-glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome. , 2010, Blood.

[12]  M. Khamashta,et al.  Antiphospholipid syndrome: pathogenesis and a window of treatment opportunities in the future , 2010, European journal of clinical investigation.

[13]  Urška Žager,et al.  ALTERATION OF ANTIBODY SPECIFICITY DURING ISOLATION AND STORAGE , 2009, Journal of immunoassay & immunochemistry.

[14]  K. Mertens,et al.  Mechanisms of Disease: antiphospholipid antibodies—from clinical association to pathologic mechanism , 2008, Nature Clinical Practice Rheumatology.

[15]  Robert L Sah,et al.  Boundary lubrication of articular cartilage: role of synovial fluid constituents. , 2007, Arthritis and rheumatism.

[16]  P. D. de Groot,et al.  High‐avidity anti‐β2 glycoprotein I antibodies highly correlate with thrombosis in contrast to low‐avidity anti‐β2 glycoprotein I antibodies , 2006 .

[17]  P. D. de Groot,et al.  Pathogenic anti-beta2-glycoprotein I antibodies recognize domain I of beta2-glycoprotein I only after a conformational change. , 2006, Blood.

[18]  C. Feighery,et al.  Circulating levels of β2-glycoprotein I in thrombotic disorders and in inflammation , 2006, Lupus.

[19]  Y. Shoenfeld,et al.  International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS) , 2006, Journal of thrombosis and haemostasis : JTH.

[20]  M. Cerinic,et al.  Accelerated Atherosclerosis in Autoimmune Rheumatic Diseases , 2005, Circulation.

[21]  S. Čučnik,et al.  Avidity of anti-beta-2-glycoprotein I antibodies. , 2005, Autoimmunity reviews.

[22]  S. Čučnik,et al.  Avidity of Anti‐β2‐Glycoprotein I and Thrombosis or Pregnancy Loss in Patients with Antiphospholipid Syndrome , 2005 .

[23]  S. Čučnik,et al.  High avidity anti-β2-glycoprotein I antibodies in patients with antiphospholipid syndrome , 2004, Annals of the rheumatic diseases.

[24]  S. Čučnik,et al.  Concomitant isolation of protein C inhibitor and unnicked β2-glycoprotein I , 2004, Clinical chemistry and laboratory medicine.

[25]  T. Kveder,et al.  Anti-β2-Glycoprotein I ELISA: Methodology, Determination of Cut-off Values in 434 Healthy Caucasians and Evaluation of Monoclonal Antibodies as Possible International Standards , 2000, Clinical chemistry and laboratory medicine.

[26]  K. Diederichs,et al.  Crystal structure of human β2‐glycoprotein I: implications for phospholipid binding and the antiphospholipid syndrome , 1999 .

[27]  P. Hogg,et al.  Microheterogeneity of beta-2 glycoprotein I: implications for binding to anionic phospholipids. , 1999, The Biochemical journal.

[28]  S. Krilis,et al.  Identification of a region of beta 2-glycoprotein I critical for lipid binding and anti-cardiolipin antibody cofactor activity. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[29]  F. Putnam,et al.  Complete amino acid sequence of human plasma beta 2-glycoprotein I. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[30]  E. Favaloro,et al.  Laboratory testing for the antiphospholipid syndrome: making sense of antiphospholipid antibody assays , 2011, Clinical chemistry and laboratory medicine.

[31]  R. Derksen,et al.  Pathogenic anti- β 2 -glycoprotein I antibodies recognize domain I of β 2 -glycoprotein I only after a conformational change , 2005 .

[32]  T. Kveder,et al.  Estimation of anticardiolipin antibodies, anti-beta2 glycoprotein I antibodies and lupus anticoagulant in a prospective longitudinal study of children with juvenile idiopathic arthritis. , 2002, Clinical and experimental rheumatology.

[33]  P. D. de Groot,et al.  β2-Glycoprotein I Is Proteolytically Cleaved In Vivo upon Activation of Fibrinolysis , 1999, Thrombosis and Haemostasis.

[34]  J. Lozier.  Complete amino acid sequence of human plasma β_2-glycoprotein I , 1984 .