QUANTITATIVE SANDWICH ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR HUMAN SECRETORY PROSTAGLANDIN-D SYNTHASE (BETA-TRACE)

Glutathione-independent prostaglandin D synthase is responsible for th e biosynthesis of prostaglandin D-2, an endogenous sleep-promoting sub stance, in the central nervous system of various mammals including hum ans. This enzyme is localized in the choroid plexus, leptomeninges, an d oligodendrocytes of the central nervous system and is secreted into the cerebrospinal fluid as beta-trace protein, a major constituent of human cerebrospinal fluid. Two monoclonal antibodies against human bet a-trace (1B7 and 10A3) were prepared by immunization of BALB/c mice wi th the recombinant protein expressed in Escherichia coli. Western blot analysis with human cerebrospinal fluid revealed that both 1B7 and 10 A3 antibodies were immunoreactive toward a single protein at the same position as that of the purified beta-trace (Mr=27,000). A quantitativ e sandwich enzyme-linked immunosorbent assay was constructed with thes e two monoclonal antibodies. The assay system showed a linearity in th e range from 0.06 to 4.00 ng beta-trace/well (100 mu l). The B-trace c oncentration was determined by the immunoassay to be 11.30+/-2.54 mu g /ml in human cerebrospinal fluid, 1.25+/-0.37 mu g/ml in the urine, an d 0.27+/-0.01 mu g/ml in the serum.