Introduction: In Australia, the ELISA technology is used in the National Johne's Disease Market Assurance Program for Cattle to assess herds for paratuberculosis. Currently, there are three commercially available ELISA kits that are approved for testing cattle for this disease in Australia. New batches of the ELISA kits are subjected to independent evaluation to assess reproducibility of the assay performance prior to release of a kit for diagnostic purposes.

Objectives: To validate criteria for the evaluation of new batches of three ELISA kits.

Experimental design: Three batches of each of the three ELISA kits were evaluated over a period of three years. Specificity sera from 180 cattle from a region considered as free of paratuberculosis and sensitivity sera from 40 cattle with paratuberculosis were tested following the kit manufacturer's recommendations.

Results: The average CVs of OD values within a plate (among wells), between plates and between batches of the three kits were 6.8% (range 3.73 to 9.12%), 9.3% (5.35 to 14.9%) and 13% (8.8 to 16.98%), respectively. The overall average agreement of diagnostic classification for all kits and batches was 99% (98 to 100%). The overall average specificity and sensitivity for all kits and batches were 99.75% (98.53 to 100%) and 78.6% (70.6 to 90.9%), respectively.

Conclusions: Data derived from this study was used to formulate acceptance criteria for evaluation of new batches of the ELISA kits. The high reproducibility of results warrants the use of these tests in market assurance programs to consistently assess level of Mycobacterium avium subsp.paratuberculosis infection in cattle herds.