NEOGEN's Estradiol ELISA is used for the quantitative analysis of Estradiol levels in biological fluid.

17β-Estradiol is considered the true ovarian hormone in women. It is the most potent estrogen, including Estrone and Estriol. 17β-Estradiol is responsible for the reproductive epithelia, breast growth, maturation of long bones, and development of the secondary female sex characteristics. The 17β-Estradiol level in plasma is used in assaying for fertility, amenorrhea and precocious puberty in girls.

Specifications
Analyte 17-β-Estradiol
Application Urine, Oral Fluid, Tissue Culture Supernatant, Plasma, Serum
Assay Range 0.02 - 2.0 ng/mL
Assay Sample Size 50 µL
Brand NEOGEN®
Drug Panel Hormones & Steroids
Intended Use For the determination of Estradiol in urine, oral fluid, tissue culture supernatant, plasma, or serum samples. Contact a NEOGEN representative for information on other matrices.
Microplate Size 96 well microplate
Platform ELISA
Result Type Quantitative
Species Non-Species Specific
Storage Conditions 2-8°C
Total Assay Incubation Time 90 minutes
Wavelength 650 nm or 450 nm with acid stop

Analytes Cross-Reactivity
17β-Estradiol 100.0%
Testosterone 1.0%
Estriol 0.41%
Estrone 0.10%
Dehydroepiandrosterone 0.03%
Aldosterone < 0.02%
Androstenedione < 0.02%
Corticosterone < 0.02%
Cortisol < 0.02%
Cortisone < 0.02%
Deoxycorticosterone < 0.02%
17-Hydroxyprogesterone < 0.02%
Pregnenolone < 0.02%
Progesterone < 0.02%
17α-Ethynylestradiol < 0.01%
β-Estradiol 17-(β-D-glucuronide) < 0.01%
  1. EIA Buffer
  2. Wash Buffer (10X)
  3. K-Blue Substrate
  4. Extraction Buffer (5X)
  5. Estradiol Enzyme Conjugate
  6. Estradiol Standard
  7. Estradiol Antibody Coated Microplate
  1. Deionized water
  2. Precision pipettes that range from 10 μL-1000 μL and disposable tips. NOTE: If all or several strips are to be used at one time, it is suggested that a multichannel pipette be used.
  3. Clean test tubes used to dilute the standards and conjugate.
  4. Graduated cylinders to dilute and mix wash buffer and extraction buffer.
  5. Microplate reader with 650 nm filter.
  6. Plate cover or plastic film to cover plate during incubation.

Additional Information

Calculation of Results

NEOGEN has partnered with MyAssays.com to provide an online tool for data calculations.

Follow the simple step-by-step guide to generate your data.

Calculate Results

MyAssays.com is a service of MyAssays Ltd. For questions or concerns about the service, please contact MyAssays Ltd.


Technology

ELISA Assay Theory

NEOGEN’s direct competitive ELISAs operate on the basis of competition between the horseradish peroxidase (HRP) enzyme conjugate and the analyte in the sample for a limited number of specific binding sites on the precoated microplate.

ELISA Assay Procedure

Samples, standards or calibrators are first added to the precoated antibody microplate. Next, the enzyme conjugate is added and the mixture is incubated at room temperature. During incubation, competition for binding sites on the microplate is taking place. The plate is then washed removing all unbound material. The bound enzyme conjugate is detected by the addition of a TMB based substrate.

ELISA test results may be obtained by measuring and comparing the absorbance reading of the wells of the samples against the standards with a microplate reader at 650 nm or 450 nm if acid stop is used. The extent of color development is inversely proportional to the amount of analyte in the sample or standard. For example, the absence of the analyte in the sample will result in a dark blue color, whereas the presence of the analyte will result in a light blue color or no color as the concentration of the analyte increases. If acid stop is used to halt the assay then the dark blue color will change to a dark yellow color and the light blue color to no color will change to light yellow to no color.

ELISA Assay Test Principle Procedure

  1. Plates are precoated with the antibody. The plate is ready for use. DO NOT WASH.
  2. A sample or control is added to each well. Next the drug-enzyme conjugate is added and the mixture is incubated at room temperature.
  3. Wash the plate to remove all unbound materials.
  4. The bound materials now remain in the microplate.
  5. Add TMB substrate to each well and allow the color to develop.
  6. Qualitative results are obtained by measuring the absorbance reading at 650 nm or 450 nm if acid stop is used.
AssayPrincipleIllustration_LifeScience.png

Training

Our customers’ success is our shared success. Our experts are ready to train you and your team on our solutions, so you can rest easy knowing procedures are performed properly and yield accurate results. In addition, we provide certificates upon completion of training.


Support

The following are common examples of unexpected results when running NEOGEN's ELISA kits with possible explanations.

  1. Extreme deep blue color development with samples and standards.
    1. The plate was not properly washed (3 x 300 µL) with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly and increase wash cycle to 5 x 300 µL.
    2. The enzyme conjugate concentrate was incorrectly diluted.

  2. Extremely low color development with samples and standards.
    1. The wash buffer was not diluted correctly before use.
    2. A poor quality of water was used to dilute the wash buffer. Deionized water should be used for dilutions.
    3. The enzyme conjugate concentrate was incorrectly diluted.
    4. The kit prematurely deteriorated, possibly from adverse shipping and storage conditions. Investigate the condition of the kit when it was received and how it was stored prior to use. Proper storage conditions are listed in the package insert.
    5. The kit has expired. Check the expiration dates on the test kit and reagents. No component of the kit should be used past the expiration date. Do not mix any reagents or components of one kit with the reagents or components of another kit.
    6. Contamination. Always use aseptic techniques when opening and removing reagents from vials and bottles. Keep the plate covered except when adding reagents, washing or reading. Always use different pipette tips for each reagent. When pipetting, do not allow the pipette tip to touch any of the reagents already in the well.

  3. No color development with samples and standards.
    1. Improper dilution of enzyme conjugate concentrate.
    2. The kit has expired. Check the expiration dates on the test kits and reagents. No components of the kit should be used past the expiration date. Do not mix any reagents or components of one kit with the reagents or components of another kit.
    3. Incorrect addition of kit reagents. Review assay procedure.

  4. Little to no displacement with the standard curve.
    1. Incorrect dilution of standards. Refer to the dilution scheme in the package insert.
    2. Contamination.
    3. The plate was not properly washed (3 x 300 µL) with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly and increase wash cycle to 5 x 300 µL.
    4. Standard has deteriorated prematurely. Contact your NEOGEN representative and provide them with the kit name, lot number, expiration date and your OD readings for further investigation.

  5. The standard curve performed correctly, but the known negative samples gave low color development.
    1. Samples need to be diluted or extracted to eliminate interference. Refer to the extraction procedure in the kit insert.

  6. The standard curve performed correctly, but the extracted samples gave low color development.
    1. The concentration of the analyte in the extracted sample is too high. The extracted sample will need to be diluted before running it in the assay so the sample OD reading will fit in the standard curve. When a dilution is used, the concentration determined from the standard curve must be multiplied by the dilution factor.
    2. Inadequate extraction of samples resulting in the presence of a solvent. Refer to the recommended extraction procedure in the package insert.

  7. Variability with duplicates.
    1. Inconsistent and/or inadequate pipetting technique when adding reagents. Improve pipetting technique.
    2. Inconsistent washing. The wells should be washed 3 x 300 µL with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly and increase wash cycle to 5 x 300 µL.
    3. Inadequate aspiration during washing. The wells should be dumped and tapped between each washing. Tap out excess liquid in wells before adding substrate. Add substrate immediately to the wells once the excess wash buffer has been removed. If using an automated washer, ensure the machine is aspirating correctly.
    4. Interruption during assay set-up. Have all reagents prepared before assay set-up commences. Reagent addition should be performed in a timely and accurate manner.