Determination of prostacyclin in plasma through a bioluminescent immunoassay for 6-keto-prostaglandin F1alpha: implication of dosage in patients with primary pulmonary hypertension.
This work describes a solid-phase immunoassay for 6-keto-prostaglandin F1alpha, the stable hydrolysis product of prostacyclin (prostaglandin I2). Prostacyclin, a potent vasodilator with antiplatelet and antiproliferative properties is an effective treatment for primary pulmonary hypertension and pulmonary arterial hypertension associated with scleroderma and scleroderma-like syndrome. Levels of 6-keto-prostaglandin F1alpha can be directly correlated with levels of prostacyclin. Therefore, 6-keto-prostaglandin F1alpha, has become the indicator of choice to measure prostacyclin levels. The single-step immunoassay for 6-keto-prostaglandin F1alpha reported here was developed using the bioluminescent protein aequorin as a label. Analyte-label conjugates were constructed by linking the carboxyl group of 6-keto-prostaglandin F1alpha and lysine residues of aequorin by chemical conjugation methods. The binding properties of 6-keto-prostaglandin F1alpha toward its antibody and the bioluminescent properties of aequorin were retained in the conjugate, which was then used to generate a dose-response curve for the analyte in a convenient microtiter plate format. The concentration of 6-keto-prostaglandin F1alpha after extraction from plasma showed good correlation with the concentration of 6-ketoprostaglandin F1alpha obtained without prior extraction of the same plasma sample. This measurement demonstrated that the assay allows the measurement of 6-keto-prostaglandin F1alpha directly in plasma without any pretreatment of the samples, which results in a much simpler method with a faster assay time.
Desai
Urvee A
UA
Department of Chemistry, University of Kentucky, Lexington 40506, USA.
Deo
Sapna K
SK
Hyland
Kenneth V
KV
Poon
Michael
M
Daunert
Sylvia
S
D015121:6-Ketoprostaglandin F1 alpha / Q000097:blood / Q000276:immunology; D000331:Aequorin; D000959:Antihypertensive Agents / Q000097:blood* / Q000493:pharmacokinetics; D016022:Case-Control Studies; D003429:Cross Reactions; D016903:Drug Monitoring / Q000379:methods* / Q000592:standards; D011464:Epoprostenol / Q000097:blood* / Q000276:immunology / Q000493:pharmacokinetics; D006801:Humans; D006976:Hypertension, Pulmonary / Q000188:drug therapy; D007118:Immunoassay / Q000379:methods; D008164:Luminescent Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000959:Antihypertensive Agents; D008164:Luminescent Proteins; D000331:Aequorin; D015121:6-Ketoprostaglandin F1 alpha; D011464:Epoprostenol
Non-programmatic