A highly sensitive sandwich enzyme immunoassay for human liver-specific antigen (LSA) and its forensic application

A highly sensitive and specific sandwich enzyme immunoassay for human liver-specific antigen (LSA) was developed and its forensic application using LSA as a marker for the determination of liver injury were examined. The LSA was purified from human liver by immunoaffinity chromatography. Polystyrene ball coated with affinity-purified rabbit anti-human LSA IgG was incubated with the human LSA and then with affinity-purified anti-human LSA Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl) propionic acid as a hydrogen donor. The detection limit for human LSA was 0.52 pg per assay. The serum LSA levels determined by this assay in healthy male and female adults were 1.5-1.6 ng/ml and 0.7-1.0 ng/ml, respectively. The recovery of LSA added to 5 microliters and 10 microliters serum samples was estimated to be 53.1-55.5% and 70.6-74.8%, respectively, and no difference in recovery between serum from males and females was observed. LSA antigenic activity in bloodstains containing LSA was detectable after storage for 30 days at room temperature. High levels of LSA were proved to exist in forensic samples taken from stabbed livers, and it was clearly possible to differentiate between samples from stabbed livers and those originating from other stabbed organs. These findings demonstrate that determination of LSA from forensic samples is useful for detecting liver injuries.