Antiphospolipidsyndrome (APS) is one of the most common autoimmune diseases characterized bythrombosis, fetal loss and presence of antiphospholipid antibodies. In APS research the antibodies of biggestinterest are anti-β2-glycoprotein I antibodies (Aβ2GPIA). β2-glycoprotein I (β2GPI)is a plasma protein which becomes activated and obtains a open structure incontact with negative charged surface molecules such as phospholipids. Inactiveβ2GPI has a closed, circular shape which can’t bind autoantibodies. Thereis no golden standard for APS diagnosing and the methods used often giveinconsistent results. The purpose of this examination project work was toconvert β2GPI into the open and closed forms, respectively, by dialyzing againsthigh ionic strength, low and high pH and determine if there is any differencein binding capacity between the two forms and Aβ2GPIAon a microtiter plate.                                                The binding capacity was tested inan ELISA (enzyme-linkedimmunosorbent assay) using purified IgG from patient sera and thedifferent conformational forms of β2GPI. An ELISA for measuring of Aβ2GPIAon several patient samples was also performed.               No difference in binding capacitycould be detected which might be explained by that the conversion of β2GPI was unsuccessful.Perhaps no difference can be measured between the structures because the closedform is expected to open on microtiter plates. An unexpected result was thepresence of immune complexes of β2GPI-Aβ2GPIA found in the serum of one of the patients. In theory an ELISA based on theopen form of β2GPI would provide more reliable diagnoses and furtherresearch is needed in this area.

The final goal of this study is to establish a microscale analysis method that allows solution phase characterization of interactions between β2-glycoprotein I (β2gpI) and some of its ligands. Human β2gpI is a phospholipid- and heparin-binding plasma glycoprotein. The physiological role of the protein in normal blood coagulation is not entirely known, nor is its role in autoimmune diseases characterized by blood clotting disturbances (thrombosis). Quantitative binding data of β2gpI interactions with some of its ligands may help elucidating the mechanisms behind these diseases and in the development of new approaches for diagnostics, prevention, and therapy. In this thesis, capillary electrophoresis (CE) was used as methodological platform for the interaction studies. The analysis of peptides and proteins by CE is desirable due to low sample consumption, possibilities for non-denaturing and highly effective separations. The first objective of this thesis was to find an approach to prevent charge dependent adsorption of β2gpI to the inner surface of the capillaries. Analyte adsorption at the negatively charged inner surface of fused silica capillaries is detrimental to interaction analyses. This phenomenon is especially pronounced in the analysis of basic proteins and proteins containing exposed positively charged domains, such as β2gpI. A new strategy to suppress these solute-wall interactions was devised, investigated and optimized. This strategy exploits the pH hysteresis behavior of fused silica surfaces, by simply performing an acidic pretreatment of the capillary. The results in this thesis show that the acidic pretreatment efficiently prevents protein adsorption. / <p>Papper 4 Estimation of the amount of β₂-glycoprotein I adsorbed at the inner surface of fused silica capillaries after acidic, neutral and alkaline pretreatment ingick som manuskript i avhandlingen, nu publicerad.</p>