A bovine monoclonal and two polyclonal conjugates, anti-llama and anti-alpaca were optimised and evaluated for a potential alpaca absorbed ELISA. The optimum conditions and hence the ability of each assay to reduce non-specific EIA reactions was measured by calculating the positive to negative ratio (P/N). The calculated P/N ratio for the anti-llama and anti-alpaca systems were 4.04 and 3.57 respectively. A P/N ratio of 5.57 was calculated for the bovine monoclonal. Based on its greater P/N ratio the bovine monoclonal ELISA was subsequently evaluated for specificity and sensitivity against five different cut-points aiming to maximise the specificity of the assay. The first two cut points were derived by the addition of 0.1 and 0.2 to the mean optical density of the specificity panel and was based on the CSL absorbed ELISA. The third and fourth cut-points required obtaining ratios by calculating the mean test optical density divided by the mean specificity panel optical density. The final cut-point involved calculating one standard deviation above the mean of the specificity panel. The specificity panel comprised of sera from 320 uninfected healthy alpaca with no history of Johne's disease. These animals were held for some time at Cocos Island quarantine station. The sensitivity panel consisted of sera from 16 alpacas with subclinical and clinical disease. A specificity of 92% and a sensitivity of 31% were obtained when the cut-point of one times the mean standard deviation of the negative sera was selected. The repeatability of the assay was assessed for variation within a plate and variation between days. The mean coefficient of variation among wells was 6.79% and between days was 12.65% for the positive and negative controls.