ELISA Kit

Catalog # KAQ0621 (96 tests)

Human
Estradiol (E2)

www.invitrogen.com
Invitrogen Corporation
542 Flynn Road, Camarillo, CA 93012
Tel: 800-955-6288
E-mail: techsupport@invitrogen.com
 Table of Contents
Table of Contents.............................................................................................................3
Contents and Storage ......................................................................................................4

Introduction....................................................................................................................5
Purpose............................................................................................................................5
Principle of the Method ....................................................................................................5
Background Information ...................................................................................................5

Methods ..........................................................................................................................6
Materials Needed But Not Provided.................................................................................6
Procedural Notes .............................................................................................................6
Directions for Washing .....................................................................................................6
Preparation of Samples and Reagents ............................................................................7
Assay Procedure..............................................................................................................8
Typical Data. ....................................................................................................................9

Performance Characteristics ......................................................................................10

Analytical Sensitivity ......................................................................................................10
Precision ........................................................................................................................10
Linearity of Dilution ........................................................................................................10
Recovery........................................................................................................................10
Antigenic Specificity .......................................................................................................11
Limitations of the Procedure ..........................................................................................11

Appendix.......................................................................................................................12
Troubleshooting Guide...................................................................................................12
Technical Support ..........................................................................................................13
Assay Summary.............................................................................................................16

PRKAQ0621 (pr111) Rev. 1.1 DCC-10-0437

 Contents and Storage

Storage Store at 2 to 8°C.

Contents

Reagents Provided Quantity Color Code

Standard 0 pg/mL in human serum, with preservatives. Lyophilized.
1 vial Yellow
Refer to vial label for quantity and reconstitution volume.
Standards 1 - 5 in human serum, with preservatives. Lyophilized.
5 vials Yellow
Refer to vial label for concentration and reconstitution volume.
Controls 1 and 2 in human serum, with preservatives. Lyophilized.
2 vials Silver
Refer to vial label for reconstitution volume and range.
Anti-Rabbit IgG-Coated Wells, 96 wells per plate. 1 plate Blue
Anti-Estradiol; lyophilized. Refer to vial label for reconstitution volume. 1 vial Blue
Estradiol-HRP Conjugate Concentrate. Contains phospate buffer with
1 vial Red
preservatives; 0.5 ml per vial.
Conjugate Buffer for dilution of Estradiol-HRP conjugate; 6 mL per vial 3 vials Red
Wash Buffer Concentrate (200x). 10 mL per bottle. 1 bottle Brown
Concentrated Chromogen, Tetramethylbenzidine (TMB) in DMF, 1 mL
1 vial Green
per vial
Substrate Buffer: H2O2 in acetate/citrate buffer; 21 mL per bottle 3 bottles White
Stop Solution; 1.8N H2SO4; 6 mL per bottle 1 bottle Black
Note: Standard 0 pg/mL is recommended for sample dilutions.

Disposal This kit contains materials with small quantities of sodium azide. Sodium azide
Note reacts with lead and copper plumbing to form explosive metal azides. Upon
disposal, flush drains with a large volume of water to prevent azide accumulation.
Avoid ingestion and contact with eyes, skin and mucous membranes. In case of
contact, rinse affected area with plenty of water. Observe all federal, state and
local regulations for disposal.

Safety All blood components and biological materials should be handled as potentially
hazardous. Follow universal precautions as established by the Centers for
Disease Control and Prevention and by the Occupational Safety and Health
Administration when handling and disposing of infectious agents.
Avoid any skin contact with H2SO4, H2O2 and TMB. In case of contact, wash
thoroughly with water.

4
 Introduction

Purpose The Invitrogen Estradiol (E2) ELISA is a competitive binding immunoassay for
the quantitative determination of 17-beta –estradiol (E2) in serum and plasma.
For Research Use Only. CAUTION: Not for human or animal therapeutic or
diagnostic use.

Principle of The Invitrogen Estradiol kit is a solid phase Enzyme Linked-Immuno-Sorbent

the Method Assay (ELISA). A fixed amount of estradiol labeled with horseradish peroxidase
(HRP) competes with unlabeled estradiol present in standards or samples for a
limited number of binding sites of a specific antibody. The E2-HRP-antibody
complex is simultaneously fixed on the wells of the microtiter plate coated with an
excess of anti-rabbit-gammaglobulins.
Neither extraction nor chromatography is required due to the high specificity of
the antibody.
After 2 hours incubation at room temperature the microtiter plate is washed to
stop the competition reaction. The chromogen solution tetramethylbenzidine
(TMB in substrate buffer) is added and incubated for 30 minutes. The reaction is
stopped with H2SO4 and the absorbance is measured at the appropriate
wavelength. The intensity of this colored product is inversely proportional to the
concentration of E2 present in the original specimen.

Background 17-beta-estradiol (E2) is a C-18 steroid hormone (molecular weight 272.4)

Information produced mainly by the ovary and placenta, and in small amounts by adrenals
and testes. Estradiol is in equilibrium with estrone, which can be converted to
estriol by the liver and placenta.

5
 Methods

Materials • Microtiter plate reader (at or near 450 nm) with software
Needed • Calibrated adjustable precision pipettes
But Not • Distilled or deionized water
Provided • Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.)
• Glass or plastic tubes for diluting solutions
• Absorbent paper towels
• Calibrated beakers and graduated cylinders

Procedural 1. When not in use, kit components should be refrigerated. All reagents should be
Notes warmed to room temperature before use.
2. Microtiter plates should be allowed to come to room temperature before
opening the foil bags. Once the desired number of strips has been removed,
immediately reseal the bag and store at 2 to 8°C to maintain plate integrity.
3. Samples should be collected in pyrogen/endotoxin-free tubes.
4. Samples should be frozen if not analyzed shortly after collection. Avoid multiple
freeze-thaw cycles of frozen samples. Thaw completely and mix well prior to
analysis.
5. When possible, avoid use of badly hemolyzed or lipemic sera. If large amounts
of particulate matter are present, centrifuge or filter prior to analysis.
6. It is recommended that all standards, controls and samples be run in duplicate.
7. When pipetting reagents, maintain a consistent order of addition from
well-to-well. This ensures equal incubation times for all wells.
8. Do not mix or interchange different reagent lots from various kit lots.
9. Do not use reagents after the kit expiration date.
10. Absorbances should be read immediately, but can be read up to 2 hours after
assay completion. For best results, keep plate covered in the dark.
11. In-house controls or kit controls, if provided, should be run with every assay. If
control values fall outside pre-established ranges, the accuracy of the assay is
suspect.
12. All residual wash liquid must be drained from the wells by efficient aspiration or
by decantation followed by tapping the plate forcefully on absorbent paper.
Never insert absorbent paper directly into the wells.
13. Because Stabilized Chromogen is light sensitive, avoid prolonged exposure to
light. Avoid contact between chromogen and metal, or color may develop.

Directions • Incomplete washing will adversely affect the test outcome. All washing
for Washing must be performed with the Wash Buffer Concentrate (200x) provided.
• Washing can be performed manually as follows: completely aspirate the liquid
from all wells by gently lowering an aspiration tip into the bottom of each well.
Take care not to scratch the inside of the well. After aspiration, fill the wells
with at least 0.4 ml of diluted Wash Buffer. Let soak for 15 to 30 seconds, then
aspirate the liquid. Repeat as directed under Assay Procedure. After the
washing procedure, the plate is inverted and tapped dry on absorbent tissue.
• Alternatively, the diluted Wash Buffer may be put into a squirt bottle. If a squirt
bottle is used, flood the plate with the diluted Wash Buffer, completely filling all
wells. After the washing procedure, the plate is inverted and tapped dry on
absorbent tissue.
• If using an automated washer, follow the washing instructions carefully.

6
 Preparation of Samples and Reagents

Sample No special pretreatment of the sample is necessary. Prior to use, all the samples
Preparation should be at room temperature. It’s recommended to vortex the samples before
use.
Do not use hemolyzed samples.
Samples may be stored for up to 72 hours at 2 to 8°C prior to testing. Samples
held for longer time should be frozen at -20°C prior to assaying.

Standards Reconstitute the lyophilized Standards and Controls to the volume specified on
and the vial label with distilled water (4 mL for the zero standard and 0.5 mL for the
other standards and controls). Allow them to remain undisturbed until completely
Controls dissolved, and then mix well by gentle inversion. See vial label for exact
concentration.
After reconstitution, store the standards and controls at 2 to 8°C for 1 week
maximum. For prolonged storage they must be frozen. Avoid freeze-thaw cycles.

Anti- Reconstitute the lyophilized Anti-Estradiol Antibody to the volume specified on the
Estradiol vial label (6 mL distilled water). Allow it to remain undisturbed until completely
dissolved, then mix well by gentle inversion.
After reconstitution, store the Anti-Estradiol at 2 to 8°C for 1 week maximum. For
prolonged storage it must be frozen. Avoid freeze-thaw cycles.

Estradiol- Pipette 0.1 mL of the concentrated HRP-estradiol solution into one of the vials of
HRP conjugate buffer. Prepare immediately prior to use.
Conjugate Maximum stability is 4 hours at room temperature or 24 hours at 2 to 8°C when
protected from direct exposure to sunlight.

Wash Dilute 2 mL of Washing Solution Concentrate in 400 mL distilled water or all the
Buffer contents of the Wash Solution Concentrate vial in 2000 mL distilled water (use a
magnetic stirrer).
The Wash Solution Concentrate is stable at room temperature until expiration
date. In order to avoid washerhead obstructions, it is recommended to prepare a
fresh diluted Wash Solution each day.

Chromogen Pipette 0.2 mL of the Concentrated Chromogen (TMB) into one of the vials of
Solution Substrate Buffer (H2O2 in acetate/citrate buffer). Prepare immediately prior to use.
Use only at room temperature. Avoid direct exposure to sunlight.
The freshly prepared Chromogen Solution is stable for a maximum of 15 min at
room temperature and must be discarded afterwards.

7
Assay Be sure to read the Procedural Notes section before carrying out the assay.
Procedure Allow all reagents to reach room temperature before use. Gently mix all liquid
reagents prior to use.
Note: A standard curve must be run with each assay.
1. Determine the number of 8-well strips needed for the assay. Insert these in
the frame for current use. (Re-bag extra strips. Store these in the refrigerator
for future use.)
2. Dispense 50 μL of each standard, control or sample into the appropriate
wells. Vertical alignment is recommended.
3. Dispense 50 μL of estradiol-HRP conjugate into all wells.
4. Dispense 50 μL of anti-estradiol into each well.
5. Incubate for 2 hours at room temperature on a horizontal shaker set at
700 ± 100 RPM.
6. Thoroughly aspirate or decant solution from wells and discard the liquid.
Wash wells 4 times. See Directions for Washing.
7. Add 200 µL of Chromogen solution to each well. The liquid in the wells will
begin to turn blue.
8. Incubate for 30 minutes at room temperature and in the dark. Note: Do
not cover the plate with aluminum foil or metalized mylar. The
incubation time for chromogen substrate is often determined by the
microtiter plate reader used. Many plate readers have the capacity to record
a maximum optical density (O.D.) of 2.0. The O.D. values should be
monitored and the substrate reaction stopped before the O.D. of the positive
wells exceeds the limits of the instrument.
9. Add 50 µL of Stop Solution to each well. Tap side of plate gently to mix. The
solution in the wells should change from blue to yellow.
10. Read the absorbance of each well at 450 nm having blanked the plate
reader against a chromogen blank composed of 100 µl each of Stabilized
Chromogen and Stop Solution. Read the plate within 1 hour after adding the
Stop Solution.
11. Use a curve fitting software to generate the standard curve. A four
parameter algorithm provides the best standard curve fit.
12. Read the concentrations for unknown samples and controls from the
standard curve.
13. For each standard and sample calculate the percent bound:
B/Bo x 100 = OD (standard or sample) x 100
OD (zero standard)
14. By interpolation of the samples (B/Bo x 100) values, determine the estradiol
concentrations of the samples from the reference curve.

8
Typical The following data were obtained for the various standards over the range of 0 to
Data 935 pg/mL E2.
(Example)
Standard Optical Density
B/Bo x 100
E2 pg/mL (450 nm)
935 0.221 12

270 0.447 25

100 0.762 43

50 1.013 57

13 1.424 80

0 1.790 100

9
 Performance Characteristics

Analytical Minimum detectable concentration (MDC) of estradiol in 10 different assays was

Sensitivity 5 ± 2 pg/mL (mean ± SD). MDC is defined as the concentration of estradiol
corresponding to 95% of maximum binding.

Precision 1. Intra-Assay Precision

Samples of known Hu Estradiol concentration were assayed in replicates of 20 to
determine precision within an assay.
Sample 1 Sample 2
Mean (pg/ml) 131 257
SD 6 10
%CV 4.6 3.9
SD = Standard Deviation
CV = Coefficient of Variation

2. Inter-Assay Precision
Samples were assayed 15 times in multiple assays to determine precision
between assays.
Sample 1 Sample 2
Mean (pg/ml) 101 196
SD 6 12
%CV 6.0 6.1
SD = Standard Deviation
CV = Coefficient of Variation

Linearity of
Theoretical Measured
Dilution Serum Recovery
conc. conc.
dilution (%)
(pg/mL) (pg/mL)
1/1 997 997 100
1/2 498 485 97
1/4 249 252 101
1/8 125 109 87 87

Recovery Added Recovery Recovery

Sample
(pg/mL) (pg/mL) (%)
Serum 916 719 78.4
516 430 83.3
316 304 96.2
166 176 106.0

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Antigenic The percentage of cross-reaction was estimated under physiological conditions in
Specificity serum by comparison of the concentration yielding a 50% binding inhibition:
Substances Cross-reactivity (%)
17-β estradiol 100
Estrone 2
Estriol 1.9
E2-3-Glucuronide 0.6
E2-17-Glucoronide 0.56
E2-17-Valerate 0.1
Cartisol <0.001
Progesterone 0.03
Dhea-sulfate <0.0001
Testosterone <0.001
Androstenediol <0.001
Norgestrel 0.01
Premarin 0.06
Equilin 0.1

Limitations Do not extrapolate the standard curve beyond the top standard point; the
of the dose-response is non-linear in this region and accuracy is difficult to obtain. Dilute
all samples above the top standard point; reanalyze these and multiply results by
Procedure the appropriate dilution factor.
The influence of various drugs, aberrant sera (hemolyzed, hyperlipidemic,
jaundiced, etc.) and the use of biological fluids in place of serum samples have
not been thoroughly investigated. The rate of degradation of native Estradiol in
various matrices has not been investigated. The immunoassay literature contains
frequent references to aberrant signals seen with some sera, attributed to
heterophilic antibodies. Though such samples have not been seen to date, the
possibility of this occurrence cannot be excluded.

11
 Appendix

Troubleshooting Guide

Elevated Cause: Insufficient washing and/or draining of wells after washing. Solution
background containing HRP-Conjugate can elevate the background if residual is left in the
well.
Solution: Wash according to the protocol. Verify the function of automated plate
washer. At the end of each washing step, invert plate on absorbent tissue on
countertop and allow to completely drain, tapping forcefully if necessary to
remove residual fluid.
Cause: Contamination of substrate solution with metal ions or oxidizing reagents.
Solution: Use distilled/deionized water for dilution of wash buffer and use plastic
equipment. DO NOT COVER plate with foil.
Cause: Contamination of pipette, dispensing reservoir or substrate solution with
HRP Conjugate.
Solution: Do not use chromogen that appears blue prior to dispensing onto the
plate. Obtain new vial of chromogen.
Cause: Incubation time is too long or incubation temperature is too high.
Solution: Reduce incubation time and/or temperature.

Elevated Cause: Incorrect dilution of standard stock solution; intermediary dilutions not
sample/ followed correctly.
standard Solution: Follow the protocol instructions regarding the dilution of the standard.
ODs Cause: Incorrect dilution of the HRP Conjugate.
Solution: Warm solution of HRP Conjugate concentrate to room temperature,
draw up slowly and wipe tip with laboratory wipe to remove excess. Dilute ONLY
in HRP diluent provided.
Cause: Incubation times extended.
Solution: Follow incubation times outlined in protocol.
Cause: Incubations carried out at 37°C when RT is dictated.
Solution: Perform incubations at RT (= 25 ± 2°C) when instructed in the protocol.

Poor Cause: Improper preparation of standard stock solution.

standard Solution: Dilute lyophilized standard as directed by the vial label only with the
curve recommended diluent buffer or in a diluent that most closely matches the matrix
of your sample.
Cause: Reagents (lyophilized standard, standard diluent buffer, etc.) from
different kits, either different cytokine or different lot number, were substituted.
Solution: NEVER substitute any components from another kit.
Cause: Errors in pipetting the standard or subsequent steps.
Solution: Always dispense into wells quickly and in the same order. Do not touch
the pipette tip on the individual microwells when dispensing. Use calibrated
pipettes and the appropriate tips for that device.

12
Weak/no Cause: Reagents not at RT (25 ± 2°C) at start of assay.
color Solution: Allow ALL reagents to warm to RT prior to commencing assay.
develops Cause: Incorrect storage of components, e.g., not stored at 2 to 8°C.
Solution: Store all components exactly as directed in protocol and on labels.
Cause: Working HRP Conjugate solution made up longer than 15 minutes before
use in assay.
Solution: Use the diluted HRP Conjugate within 15 minutes of dilution.
Cause: TMB solution lost activity.
Solution 1: The TMB solution should be clear before it is dispensed into the wells
of the microtiter plate. An intense aqua blue color indicates that the product is
contaminated. Please contact Technical Support if this problem is noted. To avoid
contamination, we recommend that the quantity required for an assay be
dispensed into a disposable trough for pipetting. Any TMB solution left in the
trough should be discarded.
Solution 2: Avoid contact of the TMB solution with items containing metal ions.
Cause: Attempt to measure analyte in a matrix for which the ELISA assay has not
been optimized.
Solution: Please contact Technical Support for advice when using alternative
sample types.
Cause: Wells have been scratched with pipette tip or washing tips.
Solution: Use caution when dispensing and aspirating into and out of microwells.

Poor Cause: Errors in pipetting the standards, samples or subsequent steps.

Precision Solution: Always dispense into wells quickly and in the same order. Do not touch
the pipette tip on the individual microwells when dispensing. Use calibrated
pipettes and the appropriate tips for that device. Check for any leaks in the pipette
tip.
Cause: Repetitive use of tips for several samples or different reagents.
Solution: Use fresh tips for each sample or reagent transfer.
Cause: Wells have been scratched with pipette tip or washing tips.
Solution: Use caution when dispensing and aspirating into and out of microwells.

Technical Support

Contact Us For more troubleshooting tips, information, or assistance, please call, email, or go
online to www.invitrogen.com/ELISA.
USA: Europe:
Invitrogen Corporation Invitrogen Ltd
542 Flynn Road Inchinnan Business Park
Camarillo, CA 93012 3 Fountain Drive
Tel: 800-955-6288 Paisley PA4 9RF, UK

E-mail: techsupport@invitrogen.com Tel: +44 (0) 141 814 6100

Fax: +44 (0) 141 814 6117
E-mail: eurotech@invitrogen.com

13
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 Explanation of symbols
Symbol Description Symbol Description

Catalogue Number Batch code

Research Use Only In vitro diagnostic medical device

Use by Temperature limitation

Manufacturer European Community authorised representative

[-] Without, does not contain [+] With, contains

Protect from light Consult accompanying documents

Directs the user to consult instructions for use (IFU), accompanying the product.

Copyright © Invitrogen Corporation. 10 February 2010

15
 Estradiol Assay Summary

SUMMARY OF ASSAY PROCEDURE

Standards (mL) Controls or samples (mL)
Standards 50 -
Controls-samples - 50
Estradiol-HRP 50 50
Anti-estradiol 50 50
Incubate for 2 hours at RT with continuous shaking (700 RPM)
Aspirate the content of each well
Wash 5 times with 0.4 mL of wash solution and aspirate
Substrate solution 200 200
Incubate 30 minutes at RT with continuous shaking (700 RPM)
H2SO4 50 50
Read the microtiter plate 450 nm (versus chromogen blank)

16