Paraneoplastic pemphigus (PNP) is a rare and severe variant of pemphigus associated with an underlying malignancy and poor therapeutic outcome. PNP is characterized by the presence of autoantibodies against a broad spectrum of desmosomal components, additionally more than 50% of patients also have antibodies against the extracellular protease inhibitor alpha 2 macroglobulin-like 1 (A2ML1). A2ML1 expression in the epidermis is restricted to the granular layer and its biological function is still unknown.
The detection of anti-A2ML1 antibodies is presently based on immunoprecipitation of differentiated human keratinocyte extracts followed by polyacrylamide gel autoradiography or immunoblotting. Faster and more reliable diagnostic tools are thus needed. Our method relies on the expression of full-length A2ML1 in fusion with enhanced green fluorescent protein (EGFP). The secreted EGFP-A2ML1 and EGFP, as negative control, were expressed in transfected human embryonic kidney 293T cells and immunoprecipitated from the culture medium with PNP A2ML1-negative or -positive sera. The measurement of fluorescence in immunoprecipitates correlates with the results obtained with current methods. Moreover, we developed an ELISA test by immobilizing EGFP-A2ML1 and EGFP on 96-well plates. With this method and the presently set cutoff value, 42 % of the tested PNP sera (n=36) were positive. Among them, 25 % were strongly positive while none of the sera of pemphigus vulgaris (n=20) and bullous pemphigoid patients (n=20) were positive.
In summary, we established efficient methods to test and quantify the reactivity of human sera against A2ML1. Similar assays may be applied for the detection of autoantibodies against other target antigens. Fluorescence immunoprecipitation is suitable for the analysis of few sera while ELISA is optimal for the screening of a large numbers of samples. Quantitative results will help to better define the phenotype(s) associated with anti-A2ML1 autoantibodies giving additional clues to the biological function of A2ML1.

Simple and sensitive assays to detect autoantibodies against alpha 2 macroglobulin-like 1 in paraneoplastic pemphigus sera