The Preparation Of β2GPI And Its Antibodies And The Preliminary Study On The Roles Of Anti-β2GPI/β2GPI

Objective:To purifyβ2-glycoprotein I(β2GPI) from normal human plasma and prepare rabbit polyclonal antibodies against humanβ2GPI for establishing the foundation of the further study on the roles of anti-β2GPI/β2GPI in the pathology of antiphospholipid syndrome(APS).Methods:1.β2GPI was purified by Heparin Sepharose CL-6B affinity chromatography following by HClO₄ precipitation.The purity of the isolatedβ2GPI was identified by SDS-PAGE,Western blotting(WB) analysis.And it's stability was observed under different temperature, concentration of salt and antiseptics.2.The New Zealand White rabbit was immunized with purified humanβ2GPI and the anti-serum was made. The whole IgG and its anti-β2GPI antibody in the anti-serum were purified by Protein A andβ2GPI-affinity column.The specificity and sensitivity of the antibody were identified by IDD,ELISA and WB.3. The monocytic cell line THP-1 cells were treated withβ2GPI and its polyclonal antibodies,and the TF activity in the cells was detected.The binding of Annexin A2 in the THP-1 cell lysates toβ2GPI was analyzed by theβ2GPI-affinity column.Results:1.β2GPI was harvested using above methods and its purity was confirmed more than 95%.The protein maintained in≤0.8 mol/L of NaCl might be degraded at room temperature,even though at 4℃.But it was not obviously degraded if it was maintained in 1.6 mol/L of NaCl at room temperature and 4℃.2.The reactive titers of the anti-serum and the whole IgG reached to 1:16 and 1:100000 with IDD and ELISA respectively.Both the whole IgG and its anti-β2GPI antibody could react withβ2GPI by WB.3.The complex ofβ2GPI and its antibody could induce the TF activity expressed on monocytic cell line THP-1.And the Annexin A2 in the THP-1 cell lysates could bind toβ2GPI-affinity column.Conclusions:1.Humanβ2GPI with high purity from the plasma can be obtained by our methods.It is important to protectβ2GPI from denaturazation in the process of conservation and regular use.It is the best way to dry the protein into powder for keepingβ2GPI long time.2. The polyclonal antibodies against humanβ2GPI can be successfully prepared by the immunization of rabbit withβ2GPI and the purification by chromatography.3.β2GPI can bind to the Annexin A2 existed in the monocytes,which has been analyzed byβ2GPI-affinity column.β2GPI and its antibodies can enhance the TF activity on monocytes,which is contributed to the mechanism of hypercoagulability in APS.