Estradiol,
17 β
–Elisa
BL-21-
E
An
Immunoenzymetric assay for the in vitro quantitative measurement of human
ESTRADIOL in serum and plasma.
For
in vitro diagnostic use only
1.
CLINICAL BACKGROUND
17
β-estradiol
(E2) is a C-18 steroid hormone (molecular weight 272.4) 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.
Like for LH, FSH, progesterone, measurement of estradiol concentration in serum,
peritoneal fluid and follicular fluid is an essential biochemical tool for the
investigation of fertility, tumor and sexual diseases, and disorders of
hypothalamic/pituitary/gonadal axis.
For example:
·
To
detect the follicular phase;
·
To
check the effectiveness of the induction of ovulation (with ultrasound) and the
level of E2 in follicular fluid. It allows normal detection or dysfunctional
ovulation induction (the empty follicle syndrome may reflect a dysfunctional
ovulation induction);
·
To
diagnose the luteinized unruptured follicle (LUF) syndrome (by the estimation of
17β
-estradiol and progesterone levels in the peritoneal fluid);
·
To
aid in the diagnosis of breast tumors (total estrogens -E1-E2- and 17β
-hydroxysteroid dehydrogenase activity are significantly higher in malignant
than in non malignant breast tissues);
·
With
LH-FSH and E2-levels, it is possible to suspect a Stein Cohen-Leventhal
syndrome;
·
Other
areas of investigation are: premature adrenarche, gynecomastie and menopausal
period
2.
PRINCIPLES OF THE METHOD
The
Bio-Line Estradiol-Elisa kit is an enzyme Immunoassay performed in microtiter
plate.
A
fixed amount of estradiol (E2) labelled with horseradish peroxydase (HRP)
competes with unlabelled estradiol present in standards or samples for a limited
number of binding sites of specific antibody (Ab.).
The
(E2-HRP-Ab.) complex is simultaneously fixed on the wells of the microtiterplate
coated with anti-rabbit-gammaglobulins in excess.
Nether
extraction nor chromatography are required due to the high specificity of the
Ab.
After
2 hours incubation at RT the microplate is washed to stop the competition
reaction.
The
revelation solution (tetramethylbenzydine (TMB)-H2O2) is added and incubated for
30 min. The reaction is stopped with H2SO4 and the microplate is read at
appropriate wavelength. The amount of substrate turnover is determined
colorimetrally by measuring the absorbance which is inversely proportional to
the estradiol concentration. A standard curve is plotted and estradiol
concentration in samples are determined by interpolation from the standard
curve.
3.
REAGENTS PROVIDED
Contents
of the Kit
1.
Strips
:12x8 (break apart) strips, 96 wells
Wells coated with anti-Rabbit IgG coated wells
2.
Anti-Estradiol:
1 vial – 6ml blue-Add 6 ml of distilled water
3.
Standards
1 to 5 + 2 controls
5 vials, 0.5 ml, lyophylised –yellow+ silver - add 0.5 ml distilled water.
See
exact values on the vial labels
4.
Standard
0:
1 vial, 1 ml, lyophylised -yellow- add 4 ml distilled water.
0 ng/ml
5.
Conjugate:
1 vial, 0.5 ml, pipet 0.1 ml into 1 vial of conjugate buffer
Estradiol-HRP conjugate in phosphate bufer
6.
conjugate
buffer :
3 vials, 6 ml, red, ready to use
7.
Chromogen
TMB:
tetramethylbenzydine ; 1 vial
1 ml green, pipett 0.2 ml into 1 vial of substrate buffer
8.
Substrate buffer: H2O2 in acetate/citrate buffer , 3 vils
White,
ready to use
9.
Stop:
1 vial, 6 ml, black, ready to use
contains 1.8 M H2SO4
Avoid contact with the stop solution. It may cause skin irritations and
burns.
10.
Wash
buffer :
1 vial, 10 ml, brown, dilute 2 ml in 400 ml dist.water
or the vial content in 2000 ml dist.water.
Note:
Zero
Calibrator is recommended for Samples
dilutions.
4.
SUPPLIES NOT PROVIDED
The
following material is required but not provided in the kit:
1.
High quality distilled water
2.
Pipettes for delivery of: 50 µl, 100 µl, 200 µl, 500 µl and 4 ml (the use of
accurate pipettes with disposable plastic tips is recommended)
3.
Vortex mixer
4.
Magnetic stirrer
5.
Horizontal microtiterplate shaker capable of 700 rpm ± 100 rpm
6.
Washer for Microtiterplates
7.
Microtiterplate reader capable of reading at 450 nm, 490 nm and 650 nm (in case
of polychromatic reading) or capable of reading at 450 nm and 650 nm
(monochromatic reading)
5.
STORAGE AND EXPIRATION DATING OF REAGENTS
·
Store
the kit at 2-8°C until the exp.date.
·
Once
opened, store the concentrated estradiol –HRP conjugate vial at 2-8°C.
Stability of diluted Estradiol conjugate is 4 hours at RT or 24h at 2-8°C.
Avoid direct exposure to sunlight.
·
After
receonstitution, store standard and controls at 2-8°C for 1 week. For prolonged
storage, these reagent have to be frozen.
·
Unused
strips wells are stored at 2-8°C in closed bag containing the dessicant until
the exp.date.
·
The
concentrate washing solution is stable at RT until the exp.date.
·
The
fresly prepared subtrate solution is stable at RT during maximum 15 minutes and
must be discarded afterwards.
6.
SPECIMEN COLLECTION AND PREPARATION
-Serum
and plasma must be kept at 2 - 8°C.
-If the test is not run within 24 hours, storage in aliquots
at -20°C is recommended. Avoid subsequent freeze thaw cycles.
-Prior to use, all samples should be at room temperature. It
is recommended to vortex the samples before use.
-Do not use haemolysed samples.
-Do
not use lipemic samples.
7.
Reagents preparation :
Standards
and controls :
Dilute
Zero standard with 4 ml of distilled water and Other standard with 0.5 ml of
distilled water.
HRP-Estradiol
conjugate :
Pipet
0.1 ml of concentrated HRP-conjugate into one vial of conjugate buffer
Stability :
24 hours at 2-8 °C avoiding sunlight.
Washing
solution :
Dilute
2 ml in 400 ml or the contents of the concentrate washing solution in 2000 ml of
distilled water.
Revelation
solution :
Pipet
0.2 ml of the chromogen TMB into one of the vials of subtrate buffer (H2O2).
Extemponaneous preparation is mandatory. Maximum stability before use : 15
min. at RT avoiding direct sunlight exposure.
8.
PROCEDURE
A.
Handling notes
Do
not use the kit or components beyond expiry date.
Do
not mix materials from different kit lots.
Bring
all the reagents to room temperature prior to use.
Thoroughly
mix all reagents and samples by gentle agitation or swirling.
Perform
calibrators, controls and samples in duplicate. Vertical alignmentis
recommended.
Use
a clean plastic container to prepare the Wash Solution.
In
order to avoid cross-contamination, use a clean disposable pipette tip for the
addition of each reagent and sample.
For
the dispensing of the Chromogenic Solution and the Stop Solution avoid pipettes
with metal parts.
High
precision pipettes or automated pipetting equipment will improve the precision.
Respect
the incubation times.
Prepare
a calibration curve for each run, do not use data from previous runs.
The
chromogenic solution should be colourless. If a dark blue colour develops within
a few minutes after preparation, this indicates that the preparation is unusable
and must be discarded.
Dispense
the Chromogenic Solution within 15 minutes following the washing of the
microtiterplate.
During
incubation with Chromogenic Solution, avoid direct sunlight on
the microtiterplate.
B.
Procedure
.
Select the required number of strips for the run.
.The
unused strips should be resealed in the bag with a desiccant and stored at 2-8°C.
.
Secure the strips into the holding frame.
1.
Add 50 µL of standards , controls and samples to designated wells.
2.
Add 50 µL of conjugate to each well.
3.
Add 50µl of anti-estradiol to each well.
4.
Incubate for 2 hours at room temperature on an orbital shaker (600-800
rpm)
5.
Aspirate and wash 4 times with 0.4 mL/well of Wash Solution.
6.
Add 200 µL of Stabilized Chromogen to each well.
7.
Incubate 30 minutes at room temperature with shaking.
8.
Add 50 µL of Stop Solution to each well.
9.
Read absorbance at 450 nm.
9.
CALCULATION OF RESULTS
Bichromatic
Reading
1.
Read the plate at 450 nm against a reference filter set at 650 nm (or 630 nm).
2.
Calculate the mean of duplicate determinations.
3.
On semi-logarithmic or linear graph paper plot the OD values (ordinate) for each
calibrator against the corresponding concentration of Estradiol (abscissa) and
draw a calibration curve through the calibrator points by connecting the plotted
points with straight lines.
4.
Read the concentration for each control and sample by interpolation on the
calibration curve.
5.
Computer assisted data reduction will simplify these calculations. If automatic
result processing is used, a 4-parameter logistic function curve fitting is
recommended.
10.
TYPICAL DATA
The
following data are for illustration only and should never be used instead ofthe
real time calibration curve.
Contents
(pg/ml)
Mean
B/Bo %
(Bichromatic model)
Std
0 0.00
1.790
100
Std
1 13
1.424
80
Std
2
50
1.013
57
Std
3 100
0.762
43
Std
4 270
0.447
25
Std
5 935
0.221
12
11.
PERFORMANCE AND LIMITATIONS
A.
Detection Limit
Twenty
zero standards were assayed along with a set of other standards.
The
detection limit, defined as the apparent concentration two standard deviations
above the average OD at zero binding, was 5 pg/ ml.
B.
Specificity
14.
PRECAUTIONS AND WARNINGS
Safety
For
in vitro diagnostic use only.
The
human blood components included in this kit have been tested by European
approved and/or FDA approved methods and found negative for HBsAg, anti-HCV,
anti-HIV-1 and 2. No known method can offer complete assurance that human blood
derivatives will not transmit hepatitis, AIDS or other infections.
Therefore,
handling of reagents, serum or plasma specimens should be in accordance with
local safety procedures.
All
animal products and derivatives have been collected from healthy animals.
Bovine
components originate from countries where BSE has not been reported.
Nevertheless,
components containing animal substances should be treated as potentially
infectious.
Avoid
any skin contact with all reagents, Stop Solution contains HCl, the chromogen
contains TMB and H2O2. In case of contact, wash thoroughly with water.
Do
not smoke, drink, eat or apply cosmetics in the working area. Do not pipette by
mouth. Use protective clothing and disposable gloves.
15.
BIBLIOGRAPHY
1.
ABRAHAM G.E. Ovarian and adrenal contribution to peripheral androgens during the
menstrual cycle. J.Clin. Endocrinol. Metab. 1974, 39:340-346
2.
BAIRD D.T. , FRASER I.S.: Blood production and ovarian secretion rates of
estradiol-17 beta and estrone in women throughout the menstrual cycle. J.Clin.
Endocrinol. Metab. 1974, 38:1009-1017
3.
HANING R.V. et al.: Maternal serum progesterone, 17-beta-estradiol and estriol
are increased in pregnanolones which follow treatment with human menopausal
gonadotropins; effects of multiple gestation and maternal endocrine status. J.
Steroid Biochem. 1985, 22:833-829
4.
MEHTA R.R. Subcellular concentrations of estrone, estradiol, androstenedione and
17b-hydroxysteroid dehydrogenase. Activity in malignant and non-malignant juman
breast tissues. Int. J. Cancer. 1987, 40:305-308
5.
SELBY et al. Dose dependent response of symptoms, pituitary and bone to
transferma oestrogen in postmenopausal women. Br. Med. J. 1986, 293:1337-1339
16.
SUMMARY OF THE PROTOCOL
CALIBRATORS(µl)
SAMPLE(S)
/ CONTROLS(µl)
Calibrators
(0-5)
50
-
Controls,
Samples
-
50
Anti-Calci-HRP
conjugate
50
50
Incubate
for 2 hours at room temperature on an orbital shaker (600-800 rpm)
Aspirate
the contents of each well.
Wash
4 times with 400 µl of Wash Solution and aspirate.
Chromogenic
Solution
200
200
Incubate
for 30 min at room temperature with continuous shaking at 700 rpm.
Stop
Solution
200
200
Read
on a microtiterplate reader and record the absorbance of each well at 450 nm
(versus
630 or 650 nm) and 490 nm (versus 630 or 650 nm)
