Basic Protocol for the ESTRAMET™ 2/16 URINARY Kit

1.    Remove white styrofoam box containing the kit reagents and the plates from the refrigerator, remove all bottles and vials from the freezer to bring all solutions to room temperature (at least one hour at R.T.).

2.    Remove urine samples from freezer and bring to room temperature. Mix and centrifuge to obtain clear sample. It is important to remove all precipitate from the samples before use, as these may interfere in the assay.

3.    Arrange microtubes in the microtube rack such that the combined 16 OHE1 and 2OHE1 standards are in triplicate (columns 1, 2, 3) for each standard in rows (B,C,D,E, and F). Keep A1, A2 and A3 wells for plate blanking (only AP Substrate is added to these wells). Arrange microtubes for positive control in wells G1-G3. Arrange triplicate tubes for each urine sample in Columns 4-12, rows A-G. This provides for 5 standards, 1 control, and 25 unknowns on each plate.

4.    Aliquot 10 microliter of each standard (0.625, 1.25, 2.5, 5.0, and 10 ng/ml of 2OHE1and 16 OHE1 combined in same standard) and urine samples into each prearranged microtube. Change pipette tips between samples. We recommend 1:4 dilution of premenopausal urine with sample diluent (1 part urine to 3 parts sample diluent).

5.    Aliquot 190 microliters of the Deconjugating Enzymes solution from a reservoir into each microtube, and mix by vortexing. This gives a 1:20 dilution of the standards and sample. Seal the tubes tightly with the adhesive plate sealer provided.

6.    Incubate (deconjugate) the samples at room temperature (20-22 deg. C) for two (2) hours. Exact temperature control is necessary for reproducibility.

7.    Neutralize the deconjugated samples after 2 hours by adding 200 microliters of the Neutralization Buffer to each microtube. Vortex to mix, and reseal if not used immediately. Samples and standards are now diluted 1:40. Deconjugated samples are best used within one-half hour.

8.    Wash the 2OHE1 and 16OHE1 EIA plates at least six times with Tris-buffered saline (TBS) pH 7.4 and 0.05% Tween-20 (see recipe section D3). TBS consist of 10 mM Tris pH 7.4 and 150 mM NaCl. Wash plate allowing each wash to remain in wells 10-15 seconds before decanting. Allow the initial wash to remain in the wells of the microtiter plate for 5 minutes before decanting liquid completely. Cover washed plates immediately with the adhesive plate cover until just before use. Use as soon as possible after washing.  Good results can not be guaranteed if plates are washed by hand.

 9.     Aliquot 75 microliters of 1:40 diluted (neutralized) standards and samples to the corresponding wells of the washed 2OHE1 and 16OHE1 plates with a multi-channel pipettor, using the same set of pipet tips for the same corresponding samples on both plates. Cover plates.

10.    Prepare 2OHE1: alkaline phosphatase conjugate solution by diluting the concentrated 2OHE1:AP Conjugate in 2OHE1 Conjugate Diluent Buffer (green-colored) to the dilution shown on the small (0.5 ml) vial of 2OHE1:AP Conjugate vial. For example, if vial cites "Use at 1:2000", Dilute 5 microliters of 2OHE1:AP Conjugate within the 10 mls of 2OHE1 Conjugate Diluent. Note : Do not eject excess contents of pipette tip- pipettors are designed to deliver exact volumes!! Prepare at least 80 microliters of diluted conjugate for each well/microtube in the final assay (e.g., about 8 mls for the whole plate. Add the diluted 2OHE1:AP conjugate to the 2OHE1 plate immediately (within 1 minute of mixing).

11.    Immediately aliquot 75 microliters of diluted 2OHE1:AP Conjugate into each assay well of the 2OHE1 assay plate. Add diluted 2OHE1:AP to 2OHE1 plate (already containing75 microliters of deconjugated-neutralized samples) within one (1) minute of conjugate dilution.

12.    Prepare 16OHE1: alkaline phosphatase conjugate solution by diluting the concentrated 16OHE1:AP Conjugate in 16OHE1 Conjugate Diluent Buffer (purple-colored) to the dilution shown on the small (0.5 ml) vial of 16OHE1:AP Conjugate vial. For example, if vial cites "Use at 1:2000", Dilute 5 microliters of 16OHE1 :AP Conjugate within the 10 ml of Conjugate Diluent. Prepare at least 80 microliters of diluted conjugate for each well/microtube in the final assay (e.g., about 8 mls for the whole plate). Apply diluted conjugate to the 16OHE1 plate immediately (within 1 minute of mixing).

13.    Immediately aliquot 75 microliters of diluted 16OHE1:AP Conjugate into each assay well of the 16OHE1 assay plate (already containing 75 microliters of deconjugated neutralized samples).

14.    Tap each assay plate several times to gently mix the contents of each well, and cover with the adhesive-coated plate cover supplied with the kit.

15.    Incubate the assay for 3 hours at room temperature (20-22 deg c). Avoid setting the covered plate in areas susceptible to drafts or temperature changes. In practice, one may incubate one assay 20 minutes longer than the other to allow reading of the plates after substrate addition at equivalent times after substrate addition. For reproducible results, however, one must perform the assay in exactly the same sequence and times, everytime.

 16.    Wash the plates 6X with TBS (pH 7.4)/0.05% Tween-20 after incubation, allowing a liquid dwell time of 10-15 seconds between washes. After the final wash, decant liquid completely by forcefully rapping inverted plate on paper towel several times. For convenience, one may stagger the washing of the 2OHE1 and 16OHE1 plates by 20 minutes, but assay sequence and times must be the same everytime.

17.   Aliquot 100 microliters of the AP Substrate (pNPP, paranitrophenyl phosphate) enzyme substrate solution to each well of the washed EIA plates with a 8-12 channel pipettor a rapidly as possible.