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Microsoft word - gd7270 00_globe_4_eng.doc
Enzyme-immunoassay for the quantitative
determination of Progesterone in serum or plasma
Progesterone is a C-21 steroid hormone involved in the
1. Reagent A – Microplate
female menstrual cycle, pregnancy and embryogenesis of
8 wells breakable strips, coated with anti-Progesterone
Progesterone is important for aldosterone
monoclonal antibody. The strips are assembled on a
(mineralocorticoid) synthesis, as 17-hydroxyprogesterone is
plastic frame and contained in a sealed bag with
desiccant. Bring the strips to room temperature before
Progesterone levels are relatively low in children and
use, to prevent any moisture formation inside the bag.
postmenopausal women. Adult males have levels similar to
those in women during the follicular phase of the menstrual
2. Reagent B – Enzymatic Tracer
In women, progesterone levels are relatively low during the
Progesterone, conjugated with Horseradish peroxidase
preovulatory phase of the menstrual cycle, rise after
ovulation, and are elevated during the luteal phase. If
pregnancy occurs, progesterone levels are maintained at
3. Reagent D/E – Chromogen/Substrate
luteal levels initially. After delivery of the placenta and
during lactation, progesterone levels are very low. The fall
Ready to use solution containing Tetramethylbenzidine
in progesterone levels following delivery is one of the
Avoid any skin contact and light exposure.
Progesterone is produced in the adrenal glands, in the
gonads (specifically after ovulation in the corpus luteum), in
4. Reagent F – Stop Solution
the brain, and, during pregnancy, in the placenta.
Progesterone converts the endometrium to its secretory
Ready to use solution containing Sulphuric acid 0.15 M.
stage to prepare the uterus for implantation.
Avoid any skin contact.
If pregnancy does not occur, progesterone levels will
decrease, leading, in the human, to menstruation.
5. Progesterone Standards:
Progesterone belongs to the group of neurosteroids that are
found in high concentrations in certain areas in the brain
Ready to use liquids containing Progesterone
and are synthesized there. Neurosteroids affect synaptic
approximately at the following concentrations:
functioning, are neuroprotective, and affect myelinization.
: 0 ng/ml, S1
: 0.2 ng/ml, S2
: 1 ng/ml, S3
: 8 ng/ml,
Progesterone is thermogenic, it reduces spasm and relaxes
: 40 ng/ml.
smooth muscle, it is involved in bronchi dilation and mucus
Actual concentrations to be used for calculation are stated
regulation. Progesterone acts as an anti inflammatory agent
Progesterone also assists in thyroid function, in bone
2 cardboard sealers to be used to cover the plate
Measurement of serum progesterone concentrations have
been used in evaluating ovarian function.
PRINCIPLE OF THE ASSAY
This test is based on “one step” competition enzyme
immunoassay principle (ELISA). Tested specimen is placed
into the microwells coated by specific anti-Progesterone-
antibodies simultaneously with Progesterone conjugated to
Horseradish peroxidase (HRP). Progesterone from the
specimen competes with the conjugated antigen for coated
antibodies. After washing procedure, the remaining
enzymatic activity bound to the microwell surface is
detected and quantified by addition of chromogen-substrate
solution. The developed colour, detected at 450 nm, is
inversely related to the quantity of Progesterone present in
Progesterone concentration in the sample is calculated
based on a series of standards.
MICROBIOLOGICAL STATE AND CLEANING
Serum or plasma (heparin, EDTA). Samples can be stored
1. All the materials of human origin resulted negative to
at 2–8 °C for a short time (max 2 day). For longer storage
HbsAg, HIV 1&2 and HCV FDA approved tests. Anyhow,
the specimen should be frozen. Avoid repeated freezing and
as no test can guarantee the absolute absence of
thawing. Highly lipemic, hemolysed or microbiologically
infective agents, handle reagents as potentially
contaminated samples should not be used in the assay.
infected, especially standards, controls and samples. All
objects come in direct contact with samples and all
residuals of the assay should be treated or eliminated
as potentially infected. Best procedures for inactivation
are treatments with autoclave at 121°C for 30 minutes
or with sodium hypochlorite at a final concentration of
A good washing procedure is essential to obtain correct and
2.5 % for 24 hours. This last method can be used for
treating the liquid washes that have to be neutralized
We therefore recommend to use a good quality ELISA
microplate washer, maintained at a good level of washing
2. Avoid any contact with skin and mucous membrane, in
Generally, 2-3 automatic washing cycles of 0.3 ml/well are
3. Use protective disposable talk-free gloves.
sufficient to avoid false positive reactions and remove high
4. Avoid contaminating reagents when taking them from
background. Anyhow we recommend to calibrate the
the vials. We recommend to use automatic pipettes
washing system on the kit itself so to match the declared
with disposable tips. When dispensing reagents, do not
touch with tips the wall of wells in order to avoid cross-
In case of manual washing, we suggest to perform 3
washing cycles, dispensing and aspirating 0.3 ml/well per
5. For the washing step, follow carefully the indications
reported in "WASHING INSTRUCTION".
In any case the liquid washed out from the plates must be
6. Avoid the substrate/chromogen to come in contact with
inactivated with a sodium hypochlorite solution at a final
oxidizing agents or metallic surfaces; avoid intense
concentration of 2.5%, before being thrown away or
light exposure during incubation or reagent
autoclaved, as it must be considered as potentially infected.
STORAGE AND STABILITY OF THE KIT
1. At least one hour before use, bring all reagents,
1. The kit has to be stored at 2-8 °C and used before the
standards and samples to room temperature (18-
30°C), mixing them carefully on vortex.
2. Unused strips have to be placed in the bag
2. Do not mix reagents from different lots.
containing the desiccant and firmly sealed before
3. We recommend to distribute standards and samples in
restore at 2-8 °C. After opening the strips are stable up
4. Distribution and incubation times must be the same for
3. All other reagents can be repeatedly used up to
exhaustion if stored at 2-8 °C, provided that they are
5. Avoid long interruptions between each step of the
handled carefully to avoid any environment
contamination. Under these conditions the reagents are
6. It is suggested to eliminate the excess of washing
stable up to the expiry date stated on the labels.
solution from the microplate after washing by blotting
7. The colour developed in the last incubation is stable
for a maximum of one hour. Otherwise, in case of
Semi automatic pipettes of 10, 200 and 1000 µl
reading after 10-15 min after dispensing stop solution,
immediately place the strips in the dark
8. We recommend to read the plate with an ELISA
automatic reader able to subtract the background at
620-630 nm and to read the absorbance of samples
Photometric reader of microplates or microstrips, linear up to at least 2 OD and supplied with filter of 450 nm
and standards at 450 nm. The "blanking" of the
instrument is to be carried out in the blank reagent
Automatic microplates washing device or manual apparatus capable of aspirating and dispensing
1. Put the desired number of microstrips into the frame.
2. If suggested analyte concentration in the sample exceeds 40 ng/ml, dilute this sample accordingly, using Standard 0.
3. Follow the scheme:
Microplate wells coated with anti-Progesterone antibody
- Cover the strips with cardboard sealer
- Incubate 60 minutes at 37 ± 1 °C
- Peel out the cardboard sealer and aspirate the reaction solution from all wells
- Rinse 3 times with 300 µl of distilled water, carefully aspirating off the remaining liquid
- Cover the strips with cardboard sealer
- Incubate 15 minutes at room temperature (22-28 °C)
, avoiding light exposure
Read the absorbance of each well against Blank at 450 nm (and 620-630 nm)
It is recommended, in each analytical run, to use control
From data obtained by Globe Diagnostics the following
sera with known Progesterone values, to check the
reference ranges are suggested. Otherwise, it is
correspondence of the obtained results with those
recommended that each laboratory establishes its own
expected and consequently validate the data.
CALCULATION OF RESULTS
1. Calculate the mean of the absorbance (Em) for each
point of the standard curve (S0 – S4) and of each
2. Plot the mean value of absorbance of the standards
(Em) against proper Progesterone concentrations.
Draw the best-fit curve through the plotted points.
3. Interpolate the values of the samples on the standard
curve to obtain the corresponding values of the
If computer controlled data reduction is used to calculate the results of the test, it is imperative that
the predicted values for the standards fall within 10%
The clinical significance of Progesterone determination can
be invalidated if the patient was treated with cortisone or
natural or synthetic steroids.
PRECAUTIONS IN USE
The reagents contain inactive components such as
The lowest detectable concentration of Progesterone is
preservatives (Sodium azide or others), surfactants etc.
The total concentration of these components is lower than
the limits reported by 67/548/EEC and 88/379/EEC
directives about classification, packaging and labelling of
dangerous substances. However, the reagents should be
Within run variation was determined by 16 replicate
handled with caution, avoiding swallowing and contact with
determination of two different control sera in the same
skin, eyes and mucous membranes. The use of laboratory
analytical run. %CV values found were < 3% according to
reagents according to good laboratory practice is
b. Inter Assay Variation
Between run variation was determined by replicate
Please refer to local legal requirements.
measurements of three different control sera in 2 different
lots. %CV values found were < 5% according to the optical
1. Wisdom, G.B. Clin. Chem. 22(8), 1255 (1976)
2. De Villa, G.O. et al J. Clin. Endoc. Metab 35, 458
The recovery of 0.2 – 1.0 – 8.0 – 40 ng/ml of Progesterone
3. Joyce, B.G., et al Steroids 29, n° 6, 761 (1977)
added to sample gave an average value (± SD) of 106% ±
4. Winkel P., et al Clin. Chem. 22(4), 422 (1976)
8.4% with reference to the original concentrations.
5. Rejkowski K.N., et al Steroids 29, n° 5, 761 (1977)
Correlation with RIA
The present kit was compared to a well-established RIA
method. 33 female sera and 30 male sera were assayed
The following linear regression curve was calculated:
The cross reaction studied and relative results are shown
The method shows no “Hook” Effect up to 200 ng/ml.
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