Characterization of anti–A2 IgG. (A) Specificity of anti–A2 IgG detected by ELISA. Lanes were loaded with either recombinant A2 (A2, 1 μg) or recombinant annexin 5 (A5, 1 μg), and IgG reactivity was analyzed by immunoblot. Positive controls were mouse monoclonal anti–human A2 IgG (MAH-A2) and rabbit polyclonal anti–human A5 (RAH-A5), respectively. Reactivity of IgG from an individual lacking anti-A2 antibodies (Healthy), and from 2 of 9 patients (D-E) positive for anti–A2 IgG is shown. Blots were developed as described in “Patients, materials, and methods” with secondary HRP-conjugated IgG. Shown are images grouped from different gels. (B) Assay of reactivity of anti-A2–containing IgG samples to other proposed plasminogen-binding proteins. SDS polyacrylamide gels were loaded with A2, actin, enolase, or p11 (1 μg/lane). Shown is reactivity of a representative patient anti-A2–containing IgG sample (patient D). Specific antibodies against the purified proteins of interest (mouse anti–human A2, rabbit anti–human actin, rabbit anti–human enolase, or mouse anti–human p11) served as positive controls (right panels). (C) Role of β2GPI. Assay of patient IgG for the presence of β2GPI (left panel). SDS polyacrylamide gels (10%) were loaded with patient purified recombinant IgG, β2GPI, or patient serum (1 μg/lane), transferred to nitrocellulose membranes, and reacted with goat anti–human β2GPI antibodies. Results indicate no detectable β2GPI in the lane containing purified IgG. Determination of assay sensitivity for β2GPI (right panel). Patient IgG (1 μg) and progressive dilutions of β2GPI (10 to 1000 ng/lane) were loaded in the same manner onto 10% SDS gels, blotted to nitrocellulose membranes, and probed with goat anti–human β2GPI antibodies. The assay was sensitive to 50 ng per lane of β2GPI. All membranes were incubated with secondary HRP-conjugated anti–goat IgG antibodies and developed.