Rapid solid-phase immunoassay for 6-keto prostaglandin F1 alpha on microplates

Abstract

We describe, for the measurement of 6-keto prostaglandin F1 alpha in biological media, a solid-phase immunoassay with immobilized antibodies that requires a total processing time of less than 2 h with hands-on time less than 30 min for 40 samples. The method combines the convenience of the microplate format with the sensitivity of radiolabeled prostaglandin derivatives as tracers in a competitive immunoassay. The intra- and interassay variations at 50% displacement of the radiolabeled prostaglandin derivative as tracer were 9.0% and 11.8%, respectively. At 50% displacement of the radiolabeled tracer, the sensitivity is about 20 pg per well. Optimal incubation time is between 60 and 90 min. Nonspecific binding was less than 1% if about 8 pg of tracer (approximately 25,000 counts/min per well) was used. Inhibition curves of samples in different dilutions were parallel to standard curves. The variation of bound radiolabeled prostaglandin derivative within the wells of one microplate (n = 96) was less than 3%. Human plasma samples and medium from tissue culture assayed for 6-keto prostaglandin F1 alpha correlated well with results obtained with a solid-phase assay based on use of magnetic particles (r = 0.99, n = 24 for culture-medium samples; r = 0.99; n = 26 for plasma samples.

Determination of Prostacyclin in Plasma through a Bioluminescent Immunoassay for 6-Keto-prostaglandin F1α: Implication of Dosage in Patients with Primary Pulmonary Hypertension

“…The detection method for prostacyclin or its breakdown product has to be extremely sensitive and accurate, since either molecule is present in very low amounts (picograms-per-milliliter) in the body. Moreover, the existing methods are time-consuming, mainly due to the necessity to extract 6-keto-PGF 1 α from biological samples. , In an effort to develop a rapid and sensitive detection method for prostacyclin, we now report a single-step, solid-phase immunoassay for 6-keto-PGF 1 α using the bioluminescent protein aequorin.…”