Estradiol ELISA 试剂盒

Allow the kit reagents to come to room temperature for 30 minutes.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Assay buffer Dilute Assay Buffer Concentrate 1:5 by adding one part of the concentrate to four parts of deionized water.
Once diluted this is stable at 4 °C for 3 months.
Wash buffer Dilute Wash Buffer Concentrate 1:20 by adding one part of the concentrate to nineteen parts of deionized water.
Once diluted this is stable at room temperature for 3 months. standard Preparation Label test tubes as #1 through #5.
Pipet 450 μL of Assay Buffer into tube #1 and 375 μL into tubes #2 to #5. the estradiol stock solution contains an organic solvent.
Prerinse the pipet tip several times to ensure accurate delivery.
Carefully add 50 μL of the estradiol stock solution to tube #1 and vortex completely.
Take 125 μL of the estradiol solution in tube #1 and add it to tube #2 and vortex completely.
Repeat the serial dilutions for tubes #3 through #5.
The concentration of estradiol in tubes 1 through 5 will be 10,000, 2,500, 625, 156.25, and 39.06 pg/mL.
Use all standards within 2 hours of preparation.

dried Fecal samples: The ethanol concentration in the final Assay Buffer dilution added to the well should be <5 % . Urine samples Urine samples should be diluted at least 1:4 times with the provided Assay Buffer. For comparison to creatinine as a urine volume marker please see our NIST-calibrated 2 plate and 10 plate Urinary Creatinine Detection kits, K002-H1 and K002-H5.

We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine estradiol concentrations.
1. Use the plate layout sheet on the back page to aid in proper sample and standard identification. Determine the number of wells to be used and return unused wells to the foil pouch with desiccant. Seal the ziploc plate bag and store at 4ºC.
2. Pipet 50 μL of samples or standards into wells in the plate.
3. Pipet 75 μL of Assay Buffer into the non-specific binding (NSB) wells.
4. Pipet 50 μL of Assay Buffer into wells to act as maximum binding wells (Bo or 0 pg/mL).
5. Add 25 μL of the DetectX® Estradiol Conjugate to each well using a repeater pipet.
6. Add 25 μL of the DetectX® Estradiol Antibody to each well, except the nsb wells, using a repeater pipet.
7. Gently tap the sides of the plate to ensure adequate mixing of the reagents. Cover the plate with the plate sealer and shake at room temperature for 2 hours. If the plate is not shaken signals bound will be approximately 20 % lower.
8. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels.
9. Add 100 μL of the TMB Substrate to each well, using a repeater pipet. 10. Incubate the plate at room temperature for 30 minutes without shaking. 11. Add 50 μL of the Stop Solution to each well, using a repeater pipet. 12. Read the optical density generated from each well in a plate reader capable of reading at 450 nm. 13. Use the plate reader's built-in 4PLC software capabilities to calculate estradiol concentration for each sample.

Average the duplicate OD readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean OD's for the NSB.
The sample concentrations obtained, calculated from the %B/B0 curve, should be multiplied by the dilution factor to obtain neat sample values.
Or Use the online tool from MyAssays to calculate the data: www.myassays.com/arbor-assays-estradiol-eia-kit.assay tyPiCAl dAtA sample Mean od net od % b/b0 Estradiol Conc. (pg/mL) NSB 0.065 0 - - Standard 1 0.110 0.045 11.0 10,000 Standard 2 0.192 0.127 27.2 2,500 Standard 3 0.359 0.294 53.6 625 Standard 4 0.614 0.549 77.2 156.25 Standard 5 0.798 0.733 94.4 39.06 B0 0.952 0.887 100.0 0 Sample 1 0.192 0.127 14.3 2,374 Sample 2 0.383 0.318 35.9 556.2 Always run your own standard curve for calculation of results. do not use this data.
Conversion Factor: 100 pg/mL of estradiol is equivalent to 367.1 pM. ® 10 EXPECT ASSAY ARTISTRY typical standard Curves 100 0.8 90 0.7 80 0.6 %B/B0 70 Net OD 0.5 60 50 0.4 %B/B0 40 0.3 30 0.2 20 0.1 10 0 0.0 10 100 1,000 10,000 Estradiol Conc. (pg/mL) Always run your own standard curves for calculation of results. do not use this data.
VAlidAtion dAtA sensitivity and limit of detection Sensitivity was calculated by comparing the OD's for twenty wells run for each of the B0 and standard #5.
The detection limit was determined at two (2) standard deviations from the B0 along the standard curve. sensitivity was determined as 39.6 pg/mL.
The Limit of Detection for the assay was determined in a similar manner by comparing the OD's for twenty runs for each of the zero standard and a low concentration human sample. limit of detection was determined as 26.5 pg/mL