Identification of human brain from a tissue fragment by detection of neurofilament proteins

We developed a method for identifying human brain from a tissue-like fragment by detection of neurofilament protein (NF) using enzyme-linked immunosorbent assay (ELISA). NF was extracted from 0.1 g of organ/tissue homogenized with Tris-HCl buffer (pH 7.2) containing urea, phenylmethylsulfonyl fluoride (PMSF), EDTA and, EGTA. It was necessary to dilute the extract at more than 2³-fold to avoid immunosuppression by urea. Positive reaction was always obtained for NF-H in 2³-fold diluted extract of brain tissue, however, NF-L and NF-M were not always detected when a brain fragment contained gray matter. Human cerebral white matter could be easily distinguished from other organs/tissues by detecting any of the NF-subunits. Brains of human and some animals could be discriminated by detecting NF-L or NF-M, although the species specificity of NF-H was not good. Our findings suggested that detection of NF-H was more useful than NF-L and NF-M for identifying a brain from a tissue-like fragment. The present ELISA method for NF-H could identify human brain specimens under the following conditions: putrefied at 4°C for up to 3 weeks, dried at 37°C for at least 4 months, heated at 50°C for at least 4 weeks. Our results showed that our method is useful for identification of brain tissue in forensic stain analysis. Two practical cases are described.