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

Figure 1

Flow chart and graphical data of   β2-glycoprotein I isolation procedure: comparison between isolation data after standard (isolation I) and optimized (isolation II) protocols. Panel A: the perchloric acid precipitation was carried out with the whole volume (V) (isolation I) and in 3 aliquots (isolation II) from the same starting material (pooled plasma from 2 AB donors, see Table 1). Panel B: elution chromatograms with NaCl gradient after heparin affinity chromatography. Panel C: elution chromatograms with NaCl gradient after cation exchange chromatography. Panel D: purity check of protein fractions collected after cationic exchange chromatography as detected by Coomassie Brilliant Blue stained 10% SDS-PAGE (~5 μg of proteins/lane). Indicated are β2GPI in 2nd and 3rd peaks of isolations I and II, respectively, and PRG4 in 1st peak after optimized protocol (isolation II). Panel E: functionality check of isolated β2GPI. Pure β2GPI was used as antigen in anti-β2GPI ELISA. Data is presented in arbitrary IgG, IgM, and IgA units—negative < 2, positive ≥ 2, and high positive ≥ 16. Legend: A.U.: absorbance units; β2GPI: β2-glycoprotein I; MM: molecular weight marker; mS/cm: millisiemens per centimeter; PRG4: proteoglycan 4; SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis.