Progesterone Safety Notes

Do not swallow the reagents. Avoid contact with eyes, skin and mucous
ELISA Test for the Quantitative Determination membranes. All patient specimens and [CAL] should be handled as
potentially infectious. [CAL] have been checked on donor level for HCV
of Progesterone in Human Serum or Plasma and HIV-1/2 antibodies and HBsAg and found negative. Wear protective
Package Size clothing and disposable gloves according to Good Laboratory Practices.
[REF] 55020 96 Tests Complete Test Kit All materials contaminated with patient specimens or [CAL] should be
[IVD] inactivated by validated procedures (autoclaving or chemical treatment)
in accordance with applicable regulations.
Intended Use [STOP] irritates eyes, skin and mucous membranes. Upon contact, rinse
Progesterone (pregn-4-ene-3, 20-dione) is a C21 steroid hormone with a thoroughly with copious amounts of water and consult a doctor.
molecular weight of 314.5 Dalton. It is the most important hormone
(progestogen) produced in non-pregnant women by the corpus luteum, in Stability
pregnant women by the placenta. Minor sources are in men the testes The reagents are stable up to the stated expiry dates on the individual
and in both sexes the adrenal cortex. labels when stored at 2...8°C.
During the menstrual cycle progesterone regulates, together with After opening reagents have to be stored at 2...8°C and used within
estrogens (estradiol), accessory organs. Furthermore it is involved in 60 days (see also "Note" ).
preparing the endometrium for the implantation of the blastocyte and in
maintaining pregnancy. [MIC]

Progesterone circulates in blood bound to binding globulins (CBG, SHBG) - Sealed in an aluminium bag with a desiccant.
and albumin. About 90-98% of the circulating hormone is bound, the - Before opening, the strips must be at room temperature.
remaining portion, the free progesterone, is assumed to be the active - Unused: return to the zip-lock bag with the desiccant. Strips stored in
steroid. this way at 2...8°C can be used for 60 days (see also "Note").
The progesterone level in blood varies widely according to the phases of - Do not touch the upper rim or the bottom of the wells with fingers.
the menstrual cycle and parallels the activity of ovarian follicle and
corpus luteum. Therefore determination of progesterone is clinically Reagent Preparation
important for confirmation of ovulation and normal function of the Bring all reagents to room temperature (15...25°C) before use.
corpus luteum in non-pregnant women1.
Reagents not in use should always be stored at 2...8°C.
Abnormal progesterone secretion2,3,4,5 may be implicated in pre-
menstrual tension, luteal insufficiency, dysmenorrhoea and irregular Working Wash Solution [WASH]
shedding of endometrium. - Dilute [WS] to 1200 ml with fresh, deionised water in a suitable
container. Rinse vial several times.
Principle - Competitive EIA -
The HUMAN PROGESTERONE ELISA is intended for professional use. The - Stability: up to 2 weeks, stored at 15...25°C.
ELISA is based on competitive interaction of progesterone and the
Specimen
hormone-enzyme conjugate for a limited number of immobilised anti-
Serum or EDTA plasma
progesterone antibodies (rabbit). Thus the amount of bound hormone-
enzyme conjugate is inversely proportional to the concentration of Do not use highly lipemic or hemolysed specimens and samples
progesterone in the specimen. containing sodium azide.
After incubation of specimen and hormone-enzyme conjugate in the well, Specimens may be stored for 3 days at 2...8°C, up to 30 days at - 20°C.
unbound conjugate is removed by washing. When substrate solution is Freeze and thaw once only. Thawed specimen must be homogenised.
added (step 2), a blue colour develops changing to yellow after stopping Eliminate particulate matter by centrifugation or filtration.
the reaction. The intensity of the colours is inversely proportional to the
amount of progesterone in the specimen. Procedure
Follow the procedure exactly as described.
The absorbance of calibrators and specimen is determined by using ELISA
microplate readers or automated ELISA systems (like HUMAN's Procedural Notes
HumaReader or ELISYS line). Concentration of un-known specimen is
P1: Do not mix or use components with different lot numbers. Do not
interpolated from a dose response curve generated by utilising serum
mix caps of vials (risk of contamination). Do not use reagents after
calibrators of known progesterone concentrations.
their expiration date.
Reagents and Contents P2: Do not use reagents that could be contaminated or look or smell
[MIC] 12 Microtiter Strips (in strip holder) different than usual.
8-well snap-off strips, coated with anti-progesterone P3: Record [CAL], specimens and controls carefully on the spread sheet
anti-bodies (rabbit) supplied with the kit.
[CAL] A-G Calibrators (white cap) P4: [MIC] - select the required number and place firmly in the holder.
7x1,0ml Ready to use, in human serum
P5: Run duplicates for [CAL], controls and specimens. Pipette them on
Progesterone - Level: 0 (A), 0.3 (B), 1.25 (C), 2.5 (D), 5.0
the bottom in the microwells.
(E), 15.0 (F) and 40.0 (G) ng/ml
P6: Always add reagents in the same order and timing to minimise
[CON] 25 ml Enzyme Antigen Conjugate (white cap)
reaction time differences between wells. This is important for
ready to use, coloured red
reproducible results. Pipetting of specimens should not exceed
Progesterone – HRP – conjugate pH 6,9 0.2
10 minutes. Otherwise pipette the calibration curve in the indicated
BSA 0.5 %
positions at half way time of the series. If more than 1 plate is used,
TRIS/MOPS buffer 0.05 mol/l
repeat the dose response curve for each plate.
NaCl 0.1 mol/l
P7: Avoid/remove air bubbles prior to incubations and reading
[WS] 30 ml Wash Solution (black cap)
absorbance.
Concentrate for ca. 1200 ml pH 7.3 0.1
TWEEN 20 0.2 % P8: [SUB] initiates and [STOP] terminates a kinetic reaction. Avoid bright
TRIS buffered saline 3.0 mmol/l light during colour development.
[SUB] 25 ml Substrate Solution (yellow cap) pH 3.5 – 4.0 P9: [MIC] - rock gently for 20-30 sec. after each pipetting step without
Ready to use spilling the solutions to ensure thorough mixing. If available mix on
3,3', 5,5'-Tetramethylbenzidin (TMB) 0.26 g/l a plate shaker.
Hydrogen peroxide 0.015 %
Sodium acetate buffer 0.05 mol/l
DMSO <5%
[STOP] 14ml Stop Solution (red cap)
Sulphuric acid 0.5 mol/l
Preservatives: Total concentration < 0.7%
Wash Procedure During pregnancy the concentration of progesterone produced by the
The wash procedure is critical. Insufficient washing will result in poor placenta increases constantly by reaching a peak value of 200 ng/ml.
precision or falsely high absorbance. Determination of the progesterone value alone is not sufficient for
W1: Aspirate off the contents, add [WASH], aspirate off after 30 sec. soak diagnosis of pathological conditions. It should be used in conjunction
time and repeat washing twice. with other clinical manifestations and diagnostic (hormone) parameters.
W2: In case of automatic washers fill and prime with [WASH]. Expected Values
Subsequently wash strips 3 times. Ensure the washer fills all wells
completely and aspirates off efficiently after 30 sec. (remaining Progesterone level
liquid: < 15 µl). Normal women:
W3: After washing, remove remaining liquid by tapping the plate upside Follicular phase 0.2 – 1.4 ng/ml
down on tissue paper. Luteal phase 4 – 25 ng/ml
Menopause 0.1 – 1 ng/ml
Pipetting Scheme
Normal men: 0.1 – 1 ng/ml
Reagents and specimens should be at room temperature before use.
Conversion factor: 1 ng/ml = 3.18 nmol/l
Step 1 Well [µl]
Each laboratory should establish its own Expected Values utilising
A1...F2 G2... instrumentation, blood collection methods and testing techniques
[CAL] A-G; in duplicate 25 -- commonly used in that laboratory.
Specimens, controls; in duplicate -- 25
Performance Characteristic
[CON] 200 200
The PROGESTERONE ELISA test has an analytic sensitivity of
Mix 0.03 - 0.07 ng/ml specimen.
Incubate 60 min at 20...25°C Specimens with progesterone concentrations above 40 ng/ml should be
Wash 3 times as described (see W1/W2 + W3) diluted (1+9) with [CAL] A (0 ng/ml) and re-assayed. Multiply the result by
[WASH] 400 400 10.
Step 2 Typical performance data can be found in the Verification Report,
[SUB] 200 200 accessible via
Incubate 15 min at 20...25°C (see P8) www.human.de/data/gb/vr/el-prog.pdf or
[STOP] 100 100 www.human-de.com/data/gb/vr/el-prog.pdf
Mix carefully Note
Measure the absorbance at 450 nm as soon as possible or within The components of the kit are stable until the expiry date even after
30 min. after termination of reaction, using a reference wavelength of opening. However, a potential contamination is directly related to the
630 - 690 nm (if available). number of samplings. The 60 days limit after first use is set for safety
reasons.
Validation of the Test
The test results are valid provided the following criteria are met: The handling should always be in compliance with common GLP
requirements (*)! The validation criteria must be met!
[CAL] Accepted range [OD]
(*This includes: Proper caps being replaced on the vials and firmly tightened / Remove only
A ≥ 1.5 reagents required for a run from stock solutions if they could come into contact with other
B < 0.93 x absorbance [CAL] A contaminating solutions like patient specimens etc. / Stock solutions always returned to
C < 0.90 x absorbance [CAL] B 2...8°C when not in use.)
D < 0.90 x absorbance [CAL] C
References
E < 0.85 x absorbance [CAL] D 1. Israel R. et al., Am. J. Obstet. Gynecol. 112, 1043 (1971)
F < 0.80 x absorbance [CAL] E
2. Soules M.R. et al., Fertil. Steril. 36, 31 (1981)
G < 0.92 x absorbance [CAL] F
3. Maganliello P.D. et al., Fertil. Steril. 36, 55 (1981)
The differences between the duplicates of [CAL] should not exceed 10%.
4. Hahlin M. et al., Hum. Reprod. 5, 622-626 (1990)
Calculation 5. Buck R.H. et al., Fertil. Steril. 50, 752-755 (1988)
Plot measured absorbances against [CAL] concentrations in a lin-lin graph.
Appropriate interpolation of plotted measuring points result in a calibra-
tion curve, from which the analyte concentration in the sample can be
EL-PROG INF 5502001 GB 12-2010-20 |
determined.
For calculation of analyte concentrations select an appropriate and vali-
dated curve fitting option (e.g. point to point).

Interpretation of Results
Progesterone concentration may vary in a single person from day to day
or even from hour to hour. Therefore serial determinations are
recommended for a proper interpretation of the results in cases for
gynaecological disorders or abnormal pregnancies.
Progesterone has a thermogenetic effect and induces an increase in basal
temperature. The interpretation of progesterone values will therefore be
easier in the context of a basal temperature curve.
The ranges of progesterone vary from less than 1 ng/ml in the follicular
phase to around 10-20 ng/ml in the luteal phase (see Expected Values).
Maximal levels are reached 6-8 days after ovulation and elevated for
4-6 days. They fall to former low levels 1-2 days before onset of
menstruation.

Human Gesellschaft für Biochemica und Diagnostica mbH