Note 1: Do not use reagents beyond the kit expiration date.

Note1 : Do not use the working substrate if it looks blue.

Ag + AbBtn AgAbBtn Note 2: Avoid extended exposure to heat and light. Opened Note 2: Do not use reagents that are contaminated or have
reagents are stable for sixty (60) days when stored at 2-8°C. bacteria growth.
Kit and component stability are identified on the label.
AbBtn = Biotinylated antibody
Note 3: Above reagents are for a single 96-well microplate. 9.0 TEST PROCEDURE
Ag = Antigen (Variable Quantity) AgAbBtn = Immune Complex
4.1 Required But Not Provided: Before proceeding with the assay, bring all reagents, serum
After a short incubation, the enzyme conjugate is added (This 1. Pipette capable of delivering 25 μl and 50 μl with a precision of references and controls to room temperature (20 - 27°C).
delayed addition permits an increase in sensitivity for low better than 1.5%. **Test Procedure should be performed by a skilled individual
concentration samples). Upon the addition of the enzyme 2. Dispenser(s) for repetitive deliveries of 0.100ml and 0.350ml or trained professional**
conjugate, competition reaction results between the enzyme analog volumes with a precision of better than 1.5%.
and the antigen in the sample for a limited number of antibody 3. Adjustable volume (200-1000µl) dispenser(s) for conjugate. 1. Format the microplates’ wells for each serum reference, control
binging sites (not consumed in the first incubation). 4. Microplate washer or a squeeze bottle (optional). and patient specimen to be assayed in duplicate. Replace any
5. Microplate Reader with 450nm and 620nm wavelength unused microwell strips back into the aluminum bag, seal
ka absorbance capability. and store at 2-8°C.
Enz Enz 6 Absorbent Paper for blotting the microplate wells. 2. Pipette 0.025 ml (25 µL) of the appropriate serum reference,
Ag + Ag + rAbBtn AgAbBtn + AgAbBtn 7 Plastic wrap or microplate cover for incubation steps. control or specimen into the assigned well.
k-a 8 Vacuum aspirator (optional) for wash steps. 3. Add 0.050 ml (50µl) of the Estradiol Biotin Reagent to all wells.
Enz
9 Timer. 4. Swirl the microplate gently for 20-30 seconds to mix.
Ag = Enzyme-antigen Conjugate (Constant Quantity) 10 Quality control materials. 5. Cover and incubate for 30 minutes at room temperature.
Estradiol (E2) Test System Enz
Ag AbBtn = Enzyme-antigen Conjugate -Antibody Complex 6. Add 0.050 ml (50µl) of Estradiol Enzyme Reagent to all wells.
Product Code: 4925-300 rAbBtn = Biotinylated antibody not reacted in first incubation 5.0 PRECAUTIONS Add directly on top the reagents dispensed in the wells .
ka = Rate Constant of Association
For In Vitro Diagnostic Use 7. Swirl the microplate gently for 20-30 seconds to mix.
k-a = Rate Constant of Disassociation Not for Internal or External Use in Humans or Animals 8. Cover and incubate for 90 minutes at room temperature.
9. Discard the contents of the microplate by decantation or
1.0 INTRODUCTION K = ka / k-a = Equilibrium Constant All products that contain human serum have been found to be non- aspiration. If decanting, blot the plate dry with absorbent paper.
reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV 10. Add 350µl of wash buffer (see Reagent Preparation Section),
A simultaneous reaction between the biotin attached to the antibody Antibodies by FDA required tests. Since no known test can offer decant (tap and blot) or aspirate. Repeat two (2) additional times
Intended Use: The Quantitative Determination of Estradiol and the streptavidin immobilized on the microwell occurs. This
Concentration in Human Serum or Plasma by a Microplate complete assurance that infectious agents are absent, all human for a total of three (3) washes. An automatic or manual plate
effects the separation of the antibody bound fraction after serum products should be handled as potentially hazardous and washer can be used. Follow the manufacturer’s instruction
Enzyme Immunoassay decantation or aspiration. capable of transmitting disease. Good laboratory procedures for for proper usage. If a squeeze bottle is employed, fill each
handling blood products can be found in the Center for Disease well by depressing the container (avoiding air bubbles) to
2.0 SUMMARY AND EXPLANATION OF THE TEST
AgAbBtn + EnzAgAbBtn + StreptavidinCW ⇒ immobilized complex Control / National Institute of Health, "Biosafety in Microbiological dispense the wash. Decant the wash and repeat two (2)
and Biomedical Laboratories," 2nd Edition, 1988, HHS Publication additional times.
Measurement of estradiol in serum or plasma is considered to be StreptavidinCW = Streptavidin immobilized on well No. (CDC) 88-8395. 11. Add 0.100 ml (100µl) of substrate solution to all wells (see
the most reliable way to assess its rate of production. Immobilized complex = sandwich complex bound to the solid Reagent Preparation Section). Always add reagents in the
surface Safe Disposal of kit components must be according to local same order to minimize reaction time differences between
Estradiol (17β-estradiol) is a steroid hormone (molecular weight of
regulatory and statutory requirement. wells.
272.3 daltons), which circulates predominantly protein-bound. In The enzyme activity in the antibody bound fraction is inversely DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION
addition to estradiol, other natural steroidal estrogens include proportional to the native antigen concentration. By utilizing several
estrone, estriol and their metabolites. Natural estrogens are 6.0 SPECIMEN COLLECTION AND PREPARATION 12. Incubate at room temperature for twenty (20) minutes.
different serum references of known antigen concentration, a dose
hormones secreted principally by the ovarian follicles and also by response curve can be generated from which the antigen 13. Add 0.050ml (50µl) of stop solution to each well and gently mix
the adrenals, corpus luteum, and placenta and, in males, by the The specimens shall be blood, serum or heparanised plasma in
concentration of an unknown can be ascertained. for 15-20 seconds. Always add reagents in the same order to
testes. Exogenous estrogens (natural or synthetic) elicit, to varying type, and taken with the usual precautions in the collection of
minimize reaction time differences between wells.
degrees, all the pharmacologic responses usually produced by venipuncture samples. For accurate comparison to establish normal
4.0 REAGENTS 14. Read the absorbance in each well at 450nm (using a reference
endogenous estrogens. values, a fasting morning serum sample should be obtained. The
wavelength of 620-630nm.. The results should be read within
blood should be collected in a redtop (with or without gel additives)
thirty (30) minutes of adding the stop solution.
Estrogenic hormones are secreted at varying rates during the
Materials Provided venipuncture tube(s) or for plasma use evacuated tube(s)
menstrual cycle throughout the period of ovarian activity. During
A. Estradiol Calibrators – 1ml/vial - Icons A-G containing heparin.. Allow the blood to clot for serum samples.
Seven (7) vials of serum reference for estradiol at Note: Dilute the samples suspected of concentrations higher than
pregnancy, the placenta becomes the main source of estrogens. At Centrifuge the specimen to separate the serum or plasma from the
concentrations of 0 (A), 20 (B), 100 (C), 250 (D), 500 (E), 1500 3000pg/ml 1:5 and 1:10 with estradiol ‘0’ pg/ml calibrator or
menopause, ovarian secretion of estrogens declines at varying cells.
male patient serum pools with a known low value for estradiol.
rates. The gonadotropins of the anterior pituitary regulate secretion (F) and 3000 (G) in pg/ml. Store at 2-8°C. A preservative has
of the ovarian hormones, estradiol and progesterone; hypothalamic been added. The calibrators can be expressed in molar Samples may be refrigerated at 2-8oC for a maximum period of five
concentrations (pM/L) by multiplying by 3.67. For example: (5) days. If the specimen(s) cannot be assayed within this time, the 10.0 CALCULATION OF RESULTS
control of pituitary gonadotropin production is in turn regulated by
plasma concentrations of the estrogens and progesterone. This 1pg/ml x 3.67= 3.67 pM/L sample(s) may be stored at temperatures of -20oC for up to 30
days. Avoid use of contaminated devices. Avoid repetitive freezing A dose response curve is used to ascertain the concentration of
complex feedback system results in the cyclic phenomenon of
B. Estradiol Enzyme Reagent – 6.0 ml/vial E and thawing. When assayed in duplicate, 0.050ml of the specimen estradiol in unknown specimens.
ovulation and menstruation.
One (1) vial of Estradiol (Analog)-horseradish peroxides (HRP) is required. 1. Record the absorbance obtained from the printout of the
conjugate in a protein-stabilizing matrix red with dye. Store at 2- microplate reader as outlined in Example 1.
Estradiol determinations have proved of value in a variety of
8°C. 7.0 QUALITY CONTROL 2. Plot the absorbance for each duplicate serum reference
contexts, including the investigation of precocious puberty in girls
versus the corresponding estradiol concentration in pg/ml on
and gynecomastia in men. Its principal uses have been in the C. Estradiol Biotin Reagent – 6.0 ml - Icon ∇ linear graph paper (do not average the duplicates of the serum
differential diagnosis of amenorrhea and in the monitoring of One (1) bottle of reagent contains anti-estradiol biotinylated Each laboratory should assay controls at levels in the low, normal
and high range for monitoring assay performance. These controls references before plotting).
ovulation induction. purified rabbit IgG conjugate in buffer, green dye and
should be treated as unknowns and values determined in every test 3. Connect the points with a best-fit curve.
preservative. Store at 2-8°C. 4. To determine the concentration of estradiol for an unknown,
This kit uses a specific anti-estradiol antibody, and does not require procedure performed. Quality control charts should be maintained
prior sample extraction of serum or plasma. Cross-reactivity to other
⇓ to follow the performance of the supplied reagents. Pertinent locate the average absorbance of the duplicates for each
D. Streptavidin Coated Plate – 96 wells –Icon unknown on the vertical axis of the graph, find the intersecting
naturally occurring and structurally related steroids is low. statistical methods should be employed to ascertain trends. The
One 96-well microplate coated with 1.0 µg/ml streptavidin and individual laboratory should set acceptable assay performance point on the curve, and read the concentration (in pg/ml) from
packaged in an aluminum bag with a drying agent. Store at limits. In addition, maximum absorbance should be consistent with the horizontal axis of the graph (the duplicates of the unknown
The employment of several serum references of known estradiol
2-8°C. past experience. Significant deviation from established performance may be averaged as indicated). In the following example, the
concentration permits construction of a graph of activity and con-
can indicate unnoticed change in experimental conditions or average absorbance (1.202) intersects the dose response
centration. From comparison to the dose response curve, an E. Wash Solution Concentrate – 20ml - Icon
degradation of kit reagents. Fresh reagents should be used to curve at (160pg/ml) estradiol concentration (See Figure 1).
unknown specimen's activity can be correlated with estradiol One (1) vial contains a surfactant in buffered saline. A
concentration. preservative has been added. Store at 2-8°C. determine the reason for the variations.
Note: Computer data reduction software designed for ELISA assay
N may also be used for the data reduction. If such software is
3.0 PRINCIPLE F. Substrate Reagent – 12ml/vial - Icon S 8.0 REAGENT PREPARATION utilized, the validation of the software should be ascertained.
One (1) bottle contains tetramethylbenzidine (TMB) and
Delayed Competitive Enzyme Immunoassay (TYPE 9): hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. 1. Wash Buffer
The essential reagents required for a enzyme immunoassay include Dilute contents of wash solution to 1000ml with distilled or
deionized water in a suitable storage container. Diluted buffer
STOP
antibody, enzyme-antigen conjugate and native antigen. G. Stop Solution – 8ml/vial - Icon
Upon mixing the biotinylated antibody with a serum containing the One (1) vial contains a strong acid(H2SO4). Store at 2-30°C. can be stored at 2-30°C for up to 60 days.
antigen, a reaction results between the antigen and the antibody.
The interaction is illustrated by the following equation: H. Product Instructions.
 EXAMPLE 1 8. Use components from the same lot. No intermixing of reagents 7. Hernandez JL, et al,”Direct evidence of luteal insufficiency in
from different batches. TABLE 3 women with habitual abortion”, Obstetric Gynecology , 49:705-
9. Accurate and precise pipetting, as well as following the exact Between Assay Precision (Values in pg/ml) 8.( 1977).

Number
Sample

Abs (A)

Abs (B)

(pg/ml)
Value
Mean
Well
time and temperature requirements prescribed are essential. 8. Goldstein D, et al, “Correlation between Estradiol and
I.D.

Any deviation from Monobind’s IFU may yield inaccurate

Sample N X σ C.V. Progesterone in cycles with luteal phase deficiency”, Fertility
results. Low 20 106.1 5.1 4.8% Sterility, 37:348-54 (1982).
10. All applicable national standards, regulations and laws, Normal 20 261.5 10.0 3.8% 9. Klopper A, Fuchs F. Progestagens. In: Fuchs F, Klopper A,
A1 2.268 including, but not limited to, good laboratory procedures, must High 20 436.7 13.5 8.2% editors. Endocrinology of Pregnancy. Hagerstown: Harper &
Cal A 2.256 0
B1 2.244 be strictly followed to ensure compliance and proper device *As measured in ten experiments in duplicate over a ten day period. Row, 99-122 (1977).
usage. 10. Lehmann F, Bettendorf G,”The endocrine shift from a normal
Cal B C1 1.839 1.849 20 11. It is important to calibrate all the equipment e.g. Pipettes, 14.2 Sensitivity cycle to anovulation”, Insler V, Bettendorf G, editors. Advances
D1 1.860 Readers, Washers and/or the automated instruments used with The estradiol AccuBind™ Microplate EIA Test System has a in Diagnosis and Treatment of Infertility. Amsterdam:
E1 1.409 this device, and to perform routine preventative maintenance. sensitivity of 8.2 pg/ml. The sensitivity was ascertained by Elseveir/North Holland, 105 -13 (1981).
Cal C 1.426 100 determining the variability of the 0 pg/ml serum calibrator and using
12. Risk Analysis- as required by CE Mark IVD Directive 98/79/EC - 11. March CM. Luteal phase defects. In: Mishell DR, Davajan V,
F1 1.443
for this and other devices, made by Monobind, can be requested the 2σ (95% certainty) statistic to calculate the minimum dose. editors. Reproductive Endocrinology, Infertility and
Cal D G1 1.017 1.003 250 via email from [email protected]. Contraception. Philadelphia: F. A. Davis Company, 1979: 469-
H1 0.989 14.3 Accuracy 76.
A2 0.698 12.2 Interpretation The Estradiol AccuBind™ Microplate ELISA Test System was 12. March CM, Goebelsmann U, Nakamura RM, Mishell Dr. Roles
Cal E 0.723 500 1. Measurements and interpretation of results must be compared with a chemiluminescence immunoassay method. of estradiol and progesterone in eliciting the midcycle luteinizing
B2 0.748 performed by a skilled individual or trained professional. Biological specimens from low, normal and relatively high estradiol hormone and follicle stimulating hormone surges. J Clin
Cal F C2 0.480 0.487 1500 2. Laboratory results alone are only one aspect for determining level populations were used (The values ranged from 10 pg/ml – Endocrinol Metab, 49:507-13 (1979).
D2 0.493 patient care and should not be the sole basis for therapy, 4300 pg/ml). The total number of such specimens was 65. The least 13. BIO-ED slide/seminar educational program. Rochester:
particularly if the results conflict with other determinants. square regression equation and the correlation coefficient were Bioeducational Publications, 1981.
E2 0.390 0.388 3000
Cal G 3. For valid test results, adequate controls and other parameters computed for this estradiol EIA in comparison with the reference 14. Radwanska E, et al,”Plasma progesterone levels in normal and
F2 0.385 must be within the listed ranges and assay requirements. method. The data obtained is displayed in Table 4. abnormal early human pregnancy”, Fertility Sterility 30:398-402
Pat# 1 G2 1.202 1.202 160 4. If test kits are altered, such as by mixing parts of different kits, TABLE 4 (1978).
H2 1.203 which could produce false test results, or if results are Least Square 15. Tietz, Textbook of clinical chemistry,2nd ed. Philadelphia:
incorrectly interpreted, Monobind shall have no liability. Mean Regression Correlation W.B. Saunders, (1994).
*The above data and table below is for example only. Do not use it 5. If computer controlled data reduction is used to interpret the Method (x) Analysis Coefficient
for calculating your results. results of the test, it is imperative that the predicted values for This 336.8 y= 36.50+1.023(x) 0.989 Revision: 4 Date: 030912 DCO: 0638
the calibrators fall within 10% of the assigned concentrations. Cat #: 4925-300
Method (y)
Reference 293.4
Figure 1 13.0 EXPECTED RANGES OF VALUES (X) Size 96(A) 192(B)
2.50
In agreement with established reference intervals for a “normal“ A) 1ml set 1ml set
Only slight amounts of bias between this method and the reference
2.00 adult population and females during gestation the expected ranges
method are indicated by the closeness of the mean values. The B) 1 (6ml) 2 (6ml)
for the Estradiol AccuBind™ ELISA Test System are detailed in
least square regression equation and correlation coefficient
Absorbance(s)

1.50 Table 1.
indicates excellent method agreement.

Reagent (fill)
C) 1 (6ml) 2 (6ml)
Patient TABLE 1
1.00 Expected Values for the Estradiol Test System
14.4 Specificity D) 1 plate 2 plates
Median Range The % cross reactivity of the estradiol antibody to selected
0.50
Females - - substances was evaluated by adding the interfering substance to a E) 1 (20ml) 1 (20ml)

Follicular Phase 48 9-175 serum matrix at various concentrations. The cross-reactivity was
0.00 F) 1 (12ml) 2 (12ml)
calculated by deriving a ratio between dose of interfering substance
Luteal Phase 103 44-196 to dose of estradiol needed to displace the same amount of labeled
0.00 0.50 1.00 1.50 2.00 2.50 3.00
G) 1 (8ml) 2 (8ml)
Periovulatory 209 107-281 analog.
Estradiol Value /1000 in pg/ml
Treated Menopausal 122 42-289
Untreated Menopausal 7.3 ND-20 Substance Cross
Note: Multiply the horizontal values by 1000 to convert into pg/ml. Reactivity
Oral Contraceptives 13 ND-103
Androstenedione 0.0003
Males 19 4-94 Dihydotestosterone 0.0008
11.0 Q.C. PARAMETERS
Cortisone <0.0001
During pregnancy the Estradiol serum levels rise rapidly till the end Corticosterone <0.0001
In order for the assay results to be considered valid the following
criteria should be met: of third trimester (17). Cortisol 0.0004
Estriol <0.0001
1. The absorbance (OD) of calibrator 0 pg/ml should be > 1.3.
It is important to keep in mind that establishment of a range of DHEA sulfate <0.0001
2. Four out of six quality control pools should be within the
values which can be expected to be found by a given method for a Estradiol <0.0001
established ranges.
population of "normal” persons is dependent upon a multiplicity of Estrone <0.0001
factors: the specificity of the method, the population tested and the Testosterone <0.0001
12.0 RISK ANALYSIS
precision of the method in the hands of the analyst. For these
reasons each laboratory should depend upon the range of expected
The MSDS and Risk Analysis Form for this product is available on
values established by the manufacturer only until an in-house range 15.0 REFERENCES
request from Monobind Inc.
can be determined by the analysts using the method with a
population indigenous to the area in which the laboratory is located. 1. Abraham GE. The application of natural steroid
12.1 Assay Performance
1. It is important that the time of reaction in each well is held radioimmunoassay to gynecologic endocrinology. In: Abraham
constant to achieve reproducible results. 14.0 PERFORMANCE CHARACTERISTICS GE, editor. Radioassay Systems in Clinical Endocrinology,
2. Pipetting of samples should not extend beyond ten (10) Basel: Marcel Dekker,: 475-529 (1981).
minutes to avoid assay drift. 14.1 Precision 2. Batzer F,”Hormonal evaluation of early pregnancy”, Fertility
3. Highly lipemic, hemolyzed or grossly contaminated The within and between assay precision of the estradiol AccuBind™ Sterility, 34:1-13 (1980).
specimen(s) should not be used. Microplate EIA Test System were determined by analyses on three 3. Bauman J, “Basal body temperature: unreliable method of
4. If more than one (1) plate is used, it is recommended to repeat different levels of pool control sera. The number, mean values, ovulation detection”, Fertility Sterility, 36:729-33, (1981).
the dose response curve. standard deviation and coefficient of variation for each of these 4. Bergquist C, Nillius SJ, Wide L “Human gonadotropin therapy:
5. The addition of substrate solution initiates a kinetic reaction, control sera are presented in Table 2 and Table 3. Serum estradiol and progesterone patterns during conceptual
which is terminated by the addition of the stop solution. cycles”, Fertility Sterility; 39:761-65 (1983).
Therefore, the substrate and stop solution should be added in TABLE 2 5. Gautray JP, et al, “Clinical investigation of the menstrual cycle:
the same sequence to eliminate any time-deviation during Within Assay Precision (Values in pg/ml) clinical, endometrial and endocrine aspects of luteal defects”,
Fertility Sterility, 35:296-303 (1981).
reaction. Sample N X σ C.V.
6. Hensleigh PA, Fainstat T, “Corpus luteum dysfunction: serum
6. Plate readers measure vertically. Do not touch the bottom of Low 20 81.9 8.1 9.9%
the wells. progesterone levels in diagnosis and assessment of therapy for
Normal 20 242.7 20.5 8.5% recurrent and threatened abortion”, Fertility Sterility, 32:396-9.
7. Failure to remove adhering solution adequately in the High 20 423.7 7.5 7.5%
aspiration or decantation wash step(s) may result in poor (1979)
replication and spurious results.