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Microsoft word - gd7270 00_globe_4_eng.docPROGESTERONE
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. S0: 0 ng/ml, S1: 0.2 ng/ml, S2: 1 ng/ml, S3: 8 ng/ml,
Progesterone is thermogenic, it reduces spasm and relaxes S4: 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 6. Cardboard
2 cardboard sealers to be used to cover the plate Measurement of serum progesterone concentrations have been used in evaluating ovarian function. 7. Package
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 REAGENTS PREPARATION
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 WASHING INSTRUCTION
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. ASSAY PROCEDURE
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 AUXILIARY MATERIALS
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 GD7270 00
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 REAGENTS Blank
- 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) QUALITY CONTROL
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 Precision
directives about classification, packaging and labelling of Variation
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 REFERENCES
1. Wisdom, G.B. Clin. Chem. 22(8), 1255 (1976) 2. De Villa, G.O. et al J. Clin. Endoc. Metab 35, 458 Recovery
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: Specificity
The cross reaction studied and relative results are shown “Hook” Effect
The method shows no “Hook” Effect up to 200 ng/ml. Manufacturer:
Via Galileo Galilei 38 - Seggiano di Pioltello (Milan) Italy
Tel: +39 02 929189 1 - Fax:+39 02 929189 39
Biology and Medicine, 1 (3): 39-43, 2009 eISSN: 09748369, www.biolmedonline.com The influence of chloroquine administration on antioxidant levels, oxidant marker and total cholesterol in Wistar rats AC Achudume Institute of Ecology and Environmental Studies, Obafemi Awolowo University, Ile-Ife, Nigeria. Abstract This study was undertaken to determine some biochemical cha