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Serological differentiation between infection and vaccination depends on the detection of pathogen specific antibodies for an epitope that is modified or lacking in a vaccine. Here we describe a new assay principle that is based on differences in the binding properties of epitope specific antibodies. C-DIVA is a potent Classical swine fever vaccine candidate that differs from the parental C-strain life attenuated vaccine in the highly immunogenic TAVSPTTLR epitope by the deletion of two and the mutation of one amino acid (TAGSΔΔTLR). We show that C-DIVA vaccination elicits antibodies with high affinity for both the TAGSΔΔTLR and TAVSPTTLR epitope, whereas infection elicits only TAVSPTTLR specific antibodies. Differentiation is achieved with a double competition assay with negative selection for antibodies with affinity for the TAGSΔΔTLR epitope followed by positive selection for antibodies with affinity for the TAVSPTTLR epitope. Our findings add a new strategy for the development of marker vaccines and their accompanying discrimination assays and offer an alternative to the devastating stamping out policy for Classical swine fever., (Copyright © 2015 Elsevier B.V. All rights reserved.)