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KMID : 1177720100040010045
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Journal of Alternatives to Animal Experiments
2010 Volume.4 No. 1 p.45 ~ p.50
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A non-radioisotopic endpoint using BrdU ELISA for Local Lymph node assay in Balb/c mice
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Jung Kyoung-Mi
Jang Won-Hee
Park Young-Ho
Lim Kyung-Min
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Abstract
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To avoid the use of radioisotopes, many efforts have been made to develop non-radioisotopic modifications of the LLNA. Recently, OECD presented a guideline for the nonradioisotopic murine local lymph node assay employing BrdU incorporation and ELISA. This method was a non-radioisotopic modification of the LLNA using 5-bromo-2′-deoxyuridine (BrdU) incorporation. However, this method recommends CBA strain mice which is not common in Korea. Here we evaluated the sensitivity of the LLNA:BrdU-ELISA using the Balb/c mice strain, which is a readily available strain in Korea. According to OECD draft guideline, female Balb/c mice were treated for 3 days with a test material or vehicle to each side of both ears and BrdU was given as a intraperitoneal administration at 5 mg/mouse. We found that Balb/c mice is sensitive to BrdU and some premature deaths were found in the treatment groups. Balb/c mice must be older than 11 weeks to tolerate BrdU. BrdU incorporation into draining lymph node cells was measured using ELISA. In this study, isoeugenol,
hexylcinnamic aldehyde(HCA), eugenol and isopropanol(IS) were used as reference chemicals. When the 1.6 or greater SI values were defined as sensitizer according to OECD draft guideline, isoeugenol, HCA and eugenol which are classified as moderate and weak sensitizers in the traditional LLNA, were found to evaluate positive results. LLNA:BrdU-ELISA using Balb/c mice, sensitivity and specificity were 100%. The EC1.6 values for isoeugenol, HCA and eugenol were calculated to be 1.8, 19.3 and 15.5% in 5 mg BrdU/mouse. A positive correlation (R2=0.813) between traditional LLNA and LLNA:BrdU-ELISA strongly suggests that the LLNA:BrdUELISA could be used as an alternative method to screen allergen.
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KEYWORD
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Local Lymph Node Assay, BrdU, ELISA, isoeugenol, HCA, eugenol, IS
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