The outcome of ELISA for antiphosphatidylethanolamine antibodies is dependent on the composition of phosphatidylethanolamine.

ABSTRACT

OBJECTIVE: The presence of circulating autoantibodies against phosphatidylethanolamine (PE) has been shown to be positively associated with symptoms of antiphospholipid syndromes (APS). However, the current ELISA-based tests for antiphosphatidylethanolamine (aPE) antibodies remain inconsistent and controversial. The term PE refers to a collection of phospholipids that have phosphorylethanolamine head group as a common structural feature, but can vary in fatty acids with diverse physicochemical properties. The present study was to investigate, using synthetic positionally symmetrical PE species as a model system, the impact of PE structural variations on aPE ELISA. METHODS: Single and combinations of synthetic PE species, including 160 (fatty acid lengthdegree of unsaturation), 180, 181, 204 and 226, were screened with ELISA using serum samples from aPE patients and compared with chicken egg PE. There were a total of 37 aPE patient serum samples, including 11 cofactor-independent IgM, 14 ABP-independent IgG and 12 ABP-dependent aPE serum samples (3 IgM, 8 IgG and 1 IgA). The ELISA conditions were investigated for different isotypes and cofactor dependence. Based on the initial screening, adjustments in phospholipid compositions were made for achieving optimal OD readings. Finally, we isolated total IgG from aPE sera to validate different antigenic preferences. RESULTS: The antigenic preference was different among immunoglobulin isotypes and between cofactor-dependent versus cofactor-independent aPE antibodies. More specifically, 181 PE was a preferred antigen for cofactor-dependent aPE, whereas 204 PE was the preferred antigen for cofactor-independent IgG aPE. In contrast, cofactor-independent IgM aPE appeared to have a general preference for a more complex PE combination with longer fatty acids and a higher degree of unsaturation. CONCLUSION: The present data indicated that the outcome of aPE ELISA was dependent on the composition and physicochemical properties of PE antigens. The discovery that aPE antibodies may have different antigenic preferences could shed light on the nature of their binding interactions.