Bovine tuberculosis (bTB) is a zoonosis mainly caused by Mycobacterium bovis . Testand-cull protocols and gross pathological examinations of abattoir animals as well as milk pasteurisation have been implemented for preventing the spread of tuberculosis from animals to humans worldwide, including in the Republic of Korea. Despite the importance of precise and rapid diagnostic tests, conventional methods, including intradermal skin tests and γ-interferon assays, are limited by the high rate of

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... se negative results for cattle in the late infectious stage as well as laborious and time-consuming procedures. Therefore, antibody detection methods, such as enzymelinked immunosorbent assay (ELISA), are urgently needed to supplement established approaches and to expand the diagnostic window. In this study, we developed a bTB ELISA by evaluating candidate recombinant and native proteins and various assay parameters. Results We produced recombinant MPB70 and SahH (rM70S) and a native 20-kDa protein (20K). The 20K ELISA showed 94.4% sensitivity and 98.2% specificity and had an optimal sample-to-positive (S/P) ratio cut-off of 0.531. rM70S ELISA showed 94.4% sensitivity and 97.3% specificity with an S/N ratio cutoff of 1.696. Conclusion The assays showed the same sensitivity but the specificity was higher for 20K ELISA than for rM70S ELISA. Both assays had acceptable diagnostic efficiency and are expected to be useful for bTB diagnosis in combination with established methods for herd screening and for expanding the diagnostic window. Background Bovine tuberculosis (bTB) is caused by Mycobacterium bovis, which can infect both humans and other taxa, including cattle [1, 2] . In South Korea, the frequencies of bTB in cattle were 0.08% (2,898 heads) at the individual level and 0.41% (427 farms) at the farm level in 2018 (https://www.kahis.go.kr/home/lkntscrinfo/selectLkntsOccrrnc.do,http://library.mafra.go.kr/skyblueimage/28195 Infected cattle are subjected to test-and-cull and compensation. Accordingly, the economic losses due to bTB in South Korea are high, in addition to the labour-intensive process of bTB diagnosis by national control agencies [3] [4] [5] . Furthermore, the potential spread from M. bovis-infected cattle to The major protein component of PPD was a 20-kDa protein, but the overall protein pattern was complex (Fig. 1) . The 20-kDa protein of PPD is a major immunoreactive protein against M. bovispositive serum. Compared with culture filtrate proteins (CFP), PPD had no proteins of larger than 20 kDa. However, the immunoreactive protein of CFP was only 20 kDa, similar to PPD. Other protein fractions from M. bovis were also analysed by SDS-PAGE and western blotting, including insoluble proteins (INS) and soluble proteins (SOL). The patterns of INS proteins were all diffuse, and the major SOL proteins were 20 and 40 kDa. The pattern of INS proteins in PPD was very different from that of CFP, while the pattern of SOL proteins was similar to that of CFP. Of INS. A 40-kDa protein showed the highest immunoreactivity by western blotting with M. bovis-positive serum. However, immunoreactive proteins were not detected in SOL. We also analysed M. avium Johnin, M. avium PPD, and M. phlei PPD. No protein bands were detected for M. avium Johnin PPD, and the major protein bands of M. phlei Statistical analysis ELISA results were analysed by Student's t-tests. All data are presented as means ± standard deviations (error bars). All statistical values were considered significant at P ≤ 0.05.