Anti-endothelial cell antibodies in systemic vasculitis and systemic lupus erythematosus (SLE): effects of heat inactivation on binding and specificity

Heating sera is used to inactivate complement but may affect the binding characteristics of autoantibodies. We studied the effect of heating sera from patients with systemic vasculitides and SLE on antibody binding to cultured human umbilical vein endothelial cells. Sera from 32 patients with systemic vasculitides, eight with SLE and 10 healthy controls were studied for anti-endothelial cell antibodies (AECA) using an ELISA before and after heating sera to 56 degrees C for 30 min. The median (range) AECA binding index in the patient group increased from 20% (0-153%) to 71.5% (10-259%) (P < 0.0001). The AECA binding index in the control group also increased from 14% (0-52%) to 90% (42-154%) (P < 0.0001). The increased binding was unaffected by the addition of fresh complement or removal of immune complexes and the increased binding after heating persisted even after cooling to 4 degrees C. Specificity experiments showed that after heating, the binding specificity of sera was lost. Removal of immunoglobulin with Protein A abolished the increased binding seen after heating. Heating sera increases AECA binding in both patient and control sera. The mechanism is probably non-specific damage to the immunoglobulin molecule, and heating sera should thus be avoided.