Ebook: Test No. 442B: Skin Sensitization : Local Lymph Node Assay: BrdU-ELISA

The Local Lymph Node Assay: BrdU-ELISA (LLNA:BrdU-ELISA) is a non-radioactive modification to the LLNA method for identifying potential skin sensitizing test substances and measuring the proliferation of lymphocytes they induce in the auricular lymph nodes. The method described in mouse (CBA/JN strain) is based on the use of measuring 5-bromo-2-deoxyuridine (BrdU) content, an analogue of thymidine, as an indicator of this proliferation. A minimum of four animals is used per dose group, with a minimum of three concentrations of the test substance, plus a concurrent negative control group and a positive control group. The experimental schedule is during 6 days. Thereafter, the animals are killed and a single cell suspension of lymph node cells (LNC) is prepared. The procedure for preparing the LNC is crucial, in particular for the small lymph nodes in NC animals. Then the BrdU content in DNA of lymphocytes is measured by ELISA using a commercial kit. This study includes: measurements (weighing, BrdU) and clinical daily observations. The results are expressed as the Stimulation Index (SI) obtained by calculation from the mean BrdU labelling index. The SI should be ⁶Ý1.6 before further evaluation of the test material as a potential skin sensitizer is warranted.  Read more...