Background: Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce GPL on the surface of the cell wall but a lipopentapeptide called L5P or ParaLP01. The molecular and genetic characterization of this antigen demonstrated that L5P is specific to MAP. Currently available diagnostic tests are based on the use of whole cell antigens that are mainly not MAP specific and that require a pre-absorption step with antigens of M. phlei. This diagnosis detect animals with a late stage infection but are not sensitive enough to detect early stage infection. We hypothesize that the pre-absorption step prevents detection of informative populations of immunoglobulins, especially in animals with subclinical faecal shedding of MAP that are not detected by the current serology-based diagnostics. The L5P antigen has now been used in various studies to evaluate a MAP specific sero-diagnostic. This synthetic molecule was used as a pure product, not requiring pre-absorption step. However a number of parameters must be assessed including the formulation of L5P and with reference to a panel of well defined serum samples.Objective: To assess the potential of L5P and its hydrosoluble variants for the serological diagnosis of MAP infection using collections of sera from different contexts.Method: In order to find the best compound for use in serology we chemically synthesized L5P and derivatives of L5P including water-soluble variants. These pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle, goats and sheep. In addition, the panels of sera included animals infected with M. bovis and M. avium subsp. hominissuis.Results: ROC analysis showed that L5P and also its water-soluble derivatives are suitable for the development of serological diagnosis. Advantageously, these pure synthetic MAP specific antigens can be produced at low cost. The use of L5P has not been validated in the contexts of ovine paratuberculosis. In the context of infections due to other mycobacteria such as M. bovis or the more closely related species M. avium subsp. hominissuis, the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.