Background: M2-type anti-mitochondrial autoantibodies are considered the hallmark of primary biliary cholangitis and are directed mainly against the E2 subunits of the 2-oxo acid dehydrogenase complex enzymes (PDC, BCOADC and OGDC). The aim of this study was to determine whether a Dot-blot that includes these E2 subunits separately could confirm the results of methods with non-separated subunits in patients with low positive or discordant results between techniques. Methods: Sera of 24 patients with low positive or discordant results and of 10 patients with clear positive results by non-separated subunits methods were analyzed by Dot-blot with separated subunits. Results: Autoantibodies against E2 subunits of PDC, BCOADC or OGDC were detected in all patients, except in one case from the low positive or discordant results group, by Dot-blot with separated subunits. Conclusions: It would be advisable to use methods that include the three E2 subunits, and a Dot-blot with separated subunits could confirm doubtful cases by non-separated assays.