Technical Abstract: ELISAs for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treating MAP bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with MAP, Mycobacterium avium subsp. avium or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2-300 seconds) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in non-specific binding by the SELISA. SELISAs prepared by treating MAP with 37% formaldehyde and then a two second burst of sonication produced the greatest difference (7X) between MAP-negative and MAP-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of MAP infections in calves experimentally inoculated with MAP or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD.