Methodology of generation and purification of anti-beta 2 glycoprotein I antibodies

In this Method Article we are showing the methodology for generation and purification of Anti-Beta 2 Glycoprotein I (β(2)GPI) antibodies. First β(2)GPI was purified from human plasma, and recognized by Western Blot and anti-β(2)GPI antibodies of serum from patients with antiphospholipid syndrome (APS). The C57BL/6 mice were immunized intraperitonealy with 150 μg of protein in adjuvant (β(2)GPI or bovine serum albumin) on days 1, 8 and 14. Then the anti-β(2)GPI antibodies were purified by affinity chromatography (Affi-Gel protein A sepharose) and affinity column using human β(2)GPI coupled to CNBr-activated Sepharose 4B. Titles of anti-β(2)GPI antibodies were determined by ELISA assays. • We purified β(2)GPI with great efficacy and that is recognized antigenically by serum from patients with SAP or an anti-β(2)gpi antibody. • We found that our purified antibody had 13 fold increased activity in ELISA test compared with the control and in Western Blot recognized with β(2)GPI (reference and purified).