Estradiol Parameter Assay Kit

Assay Procedure

1.   Prepare all reagents, standard dilutions, and samples as directed in the product insert.
2.   Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

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100 µL Estradiol Primary Antibody Solution

3.   Add 100 µL of Estradiol Primary Antibody Solution to each well (excluding the NSB wells). Cover with a plate sealer, and incubate at room temperature for 1 hour on a horizontal orbital microplate shaker.
4.   Aspirate each well and wash, repeating the process 3 times for a total of 4 washes.

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100 µL Standard, Control, or Sample

5.   Add 100 µL of Standard, control, or sample to the appropriate wells.

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100 µL Calibrator Diluent RD5-62

6.   Add 100 µL of Calibrator Diluent RD5-62 to the zero standard (B₀) wells and NSB wells.

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50 µL Estradiol Conjugate

7.   Add 50 µL of Estradiol Conjugate to all wells. Cover with a new plate sealer, and incubate at room temperature for 2 hours on the shaker.
8.   Aspirate and wash 4 times.

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200 µL Substrate Solution

9.   Add 200 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes on the benchtop. PROTECT FROM LIGHT.

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100 µL Stop Solution

10.   Add 100 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.

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