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Estradiol (human) ELISA Kit

Sensitive, Colorimetric Assay

WARNING: This product can expose you to chemicals including Estradiol, TMB, which is [are] known to the State of California to cause cancer.  For more information go to
Catalog #: K3829

Product Details

Cat # +Size K3829-100
Size 100 assays
Detection Method Absorbance (450 nm)
Species Reactivity human
Applications This ELISA kit is used for quantitative detection of Estradiol, establishing normal range.
Features & Benefits • Easy, convenient and time-saving method to assay for human Estradiol E2.
• Detection Range:2 – 50 pmol/L.
• This ELISA kit shows no species cross-reactivity.
• Cross Reactivity: No significant cross-reactivity or interference between this analyte and its analogues was observed.
Kit Components • 96 wells coated with anti-human Estradiol antibody, 1 Microplate with 2 adhesive strips
• Human Estradiol standard (72 pmol/L)
• HRP-conjugate reagent
• Standard Diluent
• Sample diluent
• Chromogen Solution A
• Chromogen Solution B
• Stop Solution
• Wash Solution (30x stock)
Storage Conditions 4°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.


Estradiol (E2 or 17β-estradiol) is the predominant female reproductive hormone secreted by the ovaries. Smaller amounts of estradiol are also produced by the adrenal cortex, and by the testes in men. BioVision’s human Estradiol E2 ELISA Kit is based on the standard principle of a sandwich enzyme-linked immunosorbent assay. Human Estradiol E2 antibody is coated on a 96-well plate. Standards and test samples are added to the wells and Estradiol E2 present in a sample is bound by the immobilized antibody. An HRP-conjugate reagent is added subsequently. After washing away the unbound antibody/HRP conjugates, HRP substrate TMB is used to visualize the HRP enzymatic reaction. TMB is catalyzed by HRP to produce a blue product that changes into yellow after adding acidic stop solution. The density of yellow color is proportional to the human Estradiol E2 captured onto the plate. This ELISA kit shows no species cross-reactivity. Detection Range: 2 – 50 pmol/L.

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This kit is listed as a Sandwich ELISA. Since estradiol is a fairly small molecule I am surprised that the assay does not need to be competitive. Can you help me understand the principle of this Sandwich assay in a situation where the molecule is probably too small to allow two antibodies to bind? Does it somehow rely on a different presentation in endogenous samples, for example, some kind of aggregate or possibly binding to sex hormone binding globulin?
For detection of small molecules, Competitive assay is usually used. But, low sensitivity is a disadvantage for Competitive ELISA as compared to Sandwich ELISA, which has better sensitivity. When molecular weight is less than 1000 kDa, the spatial structure of the antigen determines that it cannot be bound by two antibodies at the same time. However, not all Sandwich methods work this way. K3829, Estradiol ELISA Kit is based on the Open Sandwich Immunoassay (OS-ELISA) method. The principle is as follows: An antibody has a VL region and a VH region. When VL region and VH region are separated, the interaction between them will be weak without the support of the constant region (CH1/CL).The Antigen can affect the interaction between VL and VH. When the antigen binds to a variable region of the antibody, the interaction between VL and VH will be strong. It can form stable compounds. This is the principle of the Open Sandwich Immunoassay. The basic principle of Open Sandwich immunoassay is the stability of antibody variable regions that is induced by the antigen, rather than simply building a bridge between VL and VH through antigen. A special E2 antibody, developed in-house is used in this kit. We make the VL region as solid phase. VH region has HRP enzymes with special connections and is used to detect the concentration of E2 with high sensitivity. This is the general principle of the Sandwich method for K3829, Estradiol ELISA kit. The specific details are proprietary and can’t be disclosed.
What is a sandwich ELISA?
The analyte of interest in a sample is "sandwiched" by an immobilized capture antibody coated on the plate and a labeled antibody used for detection. Sandwich ELISA is very sensitive. The density of color is proportional to the amount of analyte captured from the samples and can be quantified when compared with standard curve.
What is a competitive ELISA?
The endogenous unlabled analyte of interest in a sample is "competed" by the exogenous labeled antigen coated on the plate for a limited amount of antibody binding sites. Therefore, the lower signal indicate higher concentration of the analyte.
What dilution range should I use for the samples?
A preliminary experiment is always recommended when working with new samples and ELISA Kits to ensure all results fall within the detection range.
Can I extend the standard curve (in either direction)?
Extended standard curve is not recommended by BioVision.
What type of software is needed to graph a 4-parameter or 5-parameter curve?
SoftMax Pro by Molecular Devices, SigmaPlot® by Systat Software Inc., or others can be used for this purpose.
Low absorbance; No signal
Target present below detection limits of assay
Reduce dilution factor to increase sample concentration  (Preliminary experiment is recommended for optimal dilution range)
Incompatible sample types
Use samples recommended by the kit
Standard dilutions are unstable
Use fresh standard dilutions
Buffers/substrates are contaminated
Use new buffers and substrates
Insufficient incubation time 
Extend incubation time
Cover or seal plates during all incubation 
Plate washings too vigorous
Pipette wash buffer gently or make sure the automatic wash system has correct pressure
Wells scratched with pipette or washing tips
Use caution when dispensing and aspirating into and out of wells
Incubation temperature too low
Ensure incubations are carried out at temperatures recommended by the manual
Reagents are cold
Bring all reagents to room temperature before each assay
Plate read at incorrect wavelength

Ensure the plate reader is set at the correct wavelength recommended on the protocol

High Background
Insufficient washing
Increase number of washes; Add a 30 second soaking step in between washes;  at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Residual buffer/substrates remain in wells
Remove all residual buffer and substrates at the end of each wash
Too much HRP-Streptavidin
Centrifuge and mix the substrate vial before use to avoid percipitation and inconsistent HRP concentration
Excessive incubation time
Reduce incubation time
Substrate incubation carry out in light or wait too long to read plate after adding stop solution
Avoid light during incubation and read signals immediately after adding stop solution
Standards improperly reconstituted or diluted
Briefly spin vials before opening; inspect for undissolved material after reconstitution
Poor standard curve
Standard improperly diluted
Confirm dilutions are made correctly
Standard dilutions are unstable
Use freshly diluted standards
Pipetting error 
use calibrated pipette and stay consistent between each pipette
Curve doesn't fit scale
Try plotting using different scales  (log-log, 4 parameter logistic, 5 parameter etc.)
Large intra-coefficient of variation (CV)
Bubbles in wells
Avoid bubbles during experiment, eliminate all bubbles prior to reading
Insufficient washing
Increase number of washes; add a 30 second soak step in between washes;  at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Wells not washed equally/thoroughly
Use calibrated (automated) multi-channel pipette for consistent handling, make sure all parts of the automated washer are unobstructed
Edge effects
Seal the plate completely during incubation
Incomplete reagent mixing
Make sure all reagents are mixed thoroughly during each step
Inconsistent sample preparation or storage
Consistent sample preparation. Optimize sample storage conditions and minimize freeze and thaw cycles
Stacked plates
Avoid stacking plates during incubation
Plate sealers not used or reused
Cover assay plate with plate sealer, use a fresh sealer each time to prevent contamination
Large inter-coefficient of variation (CV))
Insufficient washing
Increase number of washes; add a 30 second soak step in between washes;  at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Inconsistent incubation temperature
Make sure to follow recommended incubation temperatures. Avoid fluctuations in temperature due to environmental conditions.
Plate sealers not used or reused
Cover assay plate with plate sealer, use a fresh sealer each time to prevent contamination