Ligand blot analysis showing rTFPI ligand binds to immobilized Lp(a) and Lp(a) ligand binds to immobilized rTFPI.

(A) Purified Lp(a) at varying concentrations was subjected to nonreducing SDS-PAGE and transferred to nitrocellulose. Membranes were then incubated overnight at 4°C with rTFPI ligand. After washing, bound rTFPI ligand was identified using a primary anti-TFPI antibody and secondary antirabbit-HRP conjugate. The antigen-antibody complex was visualized using chemiluminescence. An irrelevant protein VEGF was immobilized on a separate membrane under similar conditions to Lp(a) and the membrane ligand blotted with rTFPI ligand as above. (B) rTFPI at varying concentrations electrophoresed and transferred to nitrocellulose under similar nonreducing conditions and subjected to ligand blotting with Lp(a) overnight at 4°C. Bound Lp(a) ligand was identified with primary anti-apo(a) and secondary antigoat antibodies, respectively. The antigen-antibody complex was visualized using chemiluminescence. An irrelevant protein VEGF was immobilized under similar conditions to TFPI and the membrane ligand blotted with Lp(a) as described above. C refers to a control lane where the rTFPI was omitted.