Basal level of prostaglandin D 2 in rat brain by a solid-phase enzyme immunoassay

Abstract

A solid-phase enzyme immunoassay for prostaglandin D 2 (PGD 2) was developed in which PGD 2 was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labelled and free PGD 2, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)- propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3 – 100 pg for PGD 2. The IC 50 value for PGD 2 in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enyzme immunoassay was applied to the measurement of PGD 2 content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD 2. PGD 2 contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay ( r = 0.966) after raising its contents by intravenous administration of PGD 2. The in vivo level of PGD 2 in rat brain was extremely low but determined to be 0.11 ± 0.03 ng/g tissue (mean ± S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.