6. 16a-Hydroxyestrone:Alkaline Phosphatase Conjugate (~10 ul in 500 ul/vial).

7. Positive Control Urine (300 ul)

8. Standards: 10, 5, 2.5, 1.25, and 0.625 ng/ml 2OHE1 and 16OHE1, 300 ul each combined in 1 ml vial.

9. Sample Diluent (1 ml). Use to dilute premenopausal urines 1:4 with Sample Diluent before assay, or if values are greater than 20 ng/ml. For most accurate results, if values are greater than 15ng/ml, dilute the urine sample and reassay. Values of metabolites below 1 ng/ml and above 15 ng/ml are less accurate than those on the linear portion of the standard curve.

Storage

1. PLEASE NOTE: Upon arrival of kit, please remove standards, positive control, and enzyme conjugates and store at -20 deg. C until use. Store other bottles at 2-8 deg. C. until use. Plates are kept at 2-8 deg (DO NOT FREEZE PLATES). (Store standards at -70 deg. C if not using within 2 weeks of receipt.)

2. Preparation and Storage of Samples

Urine samples are best collected with the addition of ascorbic acid to urine to prevent oxidation of labile metabolites. Fifty mg of ascorbic acid may be added to 50 ml of urine (1mg/ml). The urine should be placed at 4 deg. C immediately, labeled, and frozen at or below -20 deg. C within 8 hours. Remove any precipitate before freezing by centrifugation. Samples may be stored frozen at -20 deg. C for several months without loss of metabolites. Avoid long-term storage of frozen urine samples in frost-free refrigerators.

Devices

1. Microtubes (one box), 0.6 ml racked, 96 tubes/rack

2. Plate sealers (3 each), adhesive.

Equipment Required

1. This assay requires the use of an individual pipettor able to accurately pipet 10 ul and an 8-12 channel pipettor to pipet 75 - 200 ul/channel.

2. A multichannel manual or automatic microtiter plate washer is necessary.   Good results can not be guaranteed if plates are washed by hand.

3. RECIPE for Tris Buffer Saline (TBS) pH 7.4

10 mM Tris 1.57 grams per liter (Trizma pH 7.4, Sigma T-4003)

150 mM NaCl 8.76 grams per liter

pH to 7.4 with conc. HCl before adding Tween-20

0.05% Tween 20 0.5 ml per liter

4. Best results are obtained by reading of developed EIA plate with an automated plate reader (405 nm filter) capable of accurate endpoint and kinetic analyses of data with automatic curve fitting by four-parameter fit.

Assay Parameters

1.     The sensitivity of the 16 OHE1 assay is about 0.10 ng/ml urinary 16 OHE1 (after urine dilution of 1:40 in assay). The sensitivity of the 2OHE1 assay is about 0.1ng/ml urinary 2OHE1 (after diluted 1:40 for assay).

2.     The within-assay C.V.s for the metabolite ratio are generally less than 4% for the EIAs for 16 OHE1 and 2OHE1 assays, the between-assay C.V.s are less that 10% for the experienced operator.

3.     Both EIAs have been shown to demonstrate recovery of metabolites with serial dilution and "spiking" of exogenous estrogens into urine samples.

4.     Assay incubation time is 3 hours at room temperature, with about 30 to 60 minutes for development of alkaline phosphatase activity (color). Kinetic reading and analysis ( of slopes) of the assay is more rapid and may also be done. The entire assay requires about 6 hours to perform.

5.     Urine samples for all premenopausal women may be prediluted 1:4 (1 part urine to 3 parts diluent) with sample diluent before assay to avoid the need to reassay the sample. Multiply the resultant value by 4 to obtain the value.