Binding of recombinant human IgM anti-PC to ACs.

<p><b>A.</b> SDS-PAGE of Fab-mediated protein A purified IgM anti-PC clone p2–20 for 1 µg non-reduced (1, 3, 5) or reduced antibody (2, 4, 6). Lanes 1-2 show Coomassie brilliant blue staining, lanes 3-4 show an immunoblot staining with a conformational sensitive anti-human IgM μ-chain specific antibody (Southern Biotech) and lanes 5-6 show the membrane re-probed with a goat polyclonal anti-human IgM μ-chain specific antibody (Jackson ImmunoResearch). <b>B.</b> Flow cytometry distribution of 7AAD and Annexin V positive thymocytes after induction of apoptosis by 4 hrs incubation with dexamethasone. <b>C</b>. Analysis of the binding of IgM anti-PC clone p2–20 without sub-gating. IgM crude expression supernatant were diluted 1∶2 in 3% BSA in PBS and assessed for apoptotic cell binding. IgM p2–20 only binds to the 7AAD (+) Annexin V (+) AC population. <b>D.</b> Dose-dependent binding of IgM p2–20 to Annexin V (+) 7AAD (+) apoptotic thymocytes compared to the isotype control IgM mGO53. Binding of IgM p2–20 supernatants (<b>E</b>) or neonatal plasma pool of 4 healthy babies at 1∶25 dilution (<b>F</b>) to 7AAD (+) Annexin V (+) AC in the presence of BSA, ABA-BSA, or PC16-BSA antigens at 100 µg/ml. Binding was compared to purified isotype control myeloma IgM (Jackson ImmunoResearch) at 50 µg/ml or recombinant isotype control IgM (mGO53) at 4.5 µg/ml.