A method for detection of antibodies against the enzootic bovine leukemia virus in single serum and milk samples, as well as in pooled milk samples, is described. After the electrophoretical separation of the antigen proteins with SDS-PAGE, a transfer onto an immobilizing membrane is done, followed by immunodetection based on the reaction of the leukemia virus antibodies with antigen proteins. The antibody-antigen complex is visualized by a second, biotinylated antibody and peroxidase-avidin. This system is developed for verification of ELISA-results in surveillance of official admitted herds free of leukosis. The threshold for positive samples is tested by the European reference serum E-4, Copenhagen, the dilution 1:25,000 is recognized as positive. The detected antigen-antibody reactions are determined by scanning and comparison with the MW-markers.