Prostacyclin ELISA Kit

Sample

Tissue Culture Media, Urine

Species Reactivity

Human

Detection Method

cELISA

Intended Use

Urinary Prostacyclin ELISA Kit is a competitive immunoassay for the quantitative determination of Prostacyclin (PGI2) breakdown products in urine. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Contents of Kit

1. One 96 Well Donkey anti-Sheep IgG Plate: A plate using break-apart strips coated with donkey antibody specific to sheep IgG. 96 (8x12) wells
2. Urinary Prostacyclin Conjugate: A blue solution of alkaline phosphatase conjugated with 6-keto-PGF1α. 5 mL
3. Urinary Prostacyclin Antibody: A yellow solution of a sheep polyclonal antibody to 6k-PGF1α and 2,3d-6k-PGF1α. 5 mL
4. Assay Buffer: Tris buffered saline containing proteins and detergents and sodium azide as preservative. 27 mL
5. Wash Buffer Concentrate: Tris buffered saline containing detergents. 27 mL
6. Urinary Prostacyclin Standard: A solution of 100,000 pg/mL 2,3d-6k-PGF1α. 0.5 mL
7. p-Npp Substrate: A solution of p-nitrophenyl phosphate in buffer. Ready to use. 20 mL
8. Stop Solution: A solution of trisodium phosphate in water. Keep tightly capped. Caution: Caustic. 5 mL
9. Plate Sealer: 1 slide

Storage

All components of this kit are stable at 4°C until the kit's expiration date.

Precision

Intra-assay precision was determined by taking samples containing low, medium and high concentrations of 2,3d-6k-PGF1α and running these samples multiple times (n=24) in the same assay. Inter-assay precision was determined by measuring three samples with low, medium and high concentrations of 2,3d-6k-PGF1α in multiple assays (n=8). The precision numbers listed on the next page represent the percent coefficient of variation for the concentrations of 2,3d-6k-PGF1α determined in these assays as calculated by a 4 parameter logistic curve fitting program.

Sensitivity

Sensitivity was calculated in Assay Buffer by determining the average optical density bound for sixteen (16) wells run as Bo, and comparing to the average optical density for sixteen (16) wells run with Standard #5. The detection limit was determined as the concentration of 2,3d-6k-PGF1α measured at two (2) standard deviations from the zero along the standard curve.
Average Optical Density for the Bo = 0.731 ± 0.016 (2.18%)
Average Optical Density for Standard #5 = 0.693 ± 0.020 (2.94%)
Delta Optical Density (0-7.81 pg/mL) = 0.038
2 SD's of the Zero Standard = 2 × 0.016 = 0.032
Sensitivity = (0.032/0.038) × 7.81 pg/mL = 6.58 pg/mL

General Description

Prostacyclin (PGI2) is involved in platelet aggregation, vasoconstriction, and reproductive functions. PGI2 has a half life of 60 minutes in plasma but only 2 to 3 minutes in buffer. The production of PGI2 is typically monitored by measurement of 6k-PGF1α and 2,3d-6k-PGF1α. 6-keto-PGF1α is produced by the non-enzymatic hydration of PGI2, and is further metabolized in urine to the 2,3-dinor derivative. A number of pharmaceuticals alter and/or inhibit the synthesis of PGI2 . Methods to measure PGI2 in urine typically involve HPLC, gas chromatography/mass spectrometry, or radioimmunoassay and enzyme immunoassay for 6k-PGF1α. The conversion of PGI2 into 6k-PGF1α and 2,3d-6k-PGF1α is shown below.

Standard Curve

The results shown below are for illustration only and should not be used to calculate results.

The Prostacyclin ELISA Kit measures both 6k-PGF1α and 2,3d-6k-PGF1α in most samples. If the levels of either 6k-PGF1α or 2,3d-6k-PGF1α are to be determined independently, then the samples should be measured in other ELISA Kit. Please contact the CD technician for inquiry.
A typical standard curve is shown below. This curve must not be used to calculate Urinary Prostacyclin concentrations; each user must run a standard curve for each assay.