DiaMetra Progesterone Elisa Kit

CLINICAL SIGNIFICANCE
Progesterone is a hormone synthesised in the ovaries, adrenal glands and placenta. It diffuses into circulation where it binds to plasma steroid-binding proteins such as albumin and corticosteroid binding protein. Progesterone is then transported toward the target organs including the hypothalamus, ovaries, uterus but also the liver, where it is catabolised.
Progesterone is fundamental in the early phase of pregnancy since it is required for uterine receptivity for embryo implantation1 . During the early follicular phase of the female menstrual cycle, the majority of circulating progesterone is of adrenal production. However, in the late follicular phase, there is a rise in progesterone serum levels released by the corpus luteum, a luteinised ovarian follicle1 . This physiological increase of progesterone is crucial to ensure the secretory function of endometrium and a normal embryo implantation2 . It does so by controlling the maternal immune system to promote maternal immunotolerance to avoid embryo rejection and reduces uterine contractility and improves utero-placental circulation for proper growth of the embryo.
Measurement of progesterone is useful to aid the assessment of ovulation and to aid in the diagnosis of ovarian dysfunctions that could lead to infertility. In addition, assessment of progesterone is also used as an aid in the diagnosis of ectopic pregnancy (EP), where levels are lower in EP than intrauterine pregnancy.
Progesterone levels are relatively low in children and postmenopausal women. Adult males have levels similar to those in women during the follicular phase of the menstrual cycle.

PRINCIPLE OF THE METHOD
The Progesterone ELISA is a competitive enzyme immunometric assay (ELISA) where progesterone (antigen) in the sample competes with the antigenic progesterone conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti progesterone coated on the microplate (solid phase).
After the incubation, the bound/free separation is performed by a simple solid phase washing. Then, the enzyme HRP in the bound fraction reacts with the Substrate (H2O2) and the TMB Substrate and develops a blue colour that changes into yellow when the Stop Solution (H2SO4) is added. The colour intensity is inversely proportional to the progesterone concentration in the sample.
Progesterone concentration in the sample is calculated through a calibration curve.

REAGENT STORAGE AND STABILITY
Store the kit at 2 – 8°C in the dark. 
The kit is stable at 2 – 8°C until the expiry date stated on the external kit label.
Once opened, the kit is stable at 2 – 8°C for 6 months. 
The diluted wash solution is stable for 30 days at 2-8°C.
Important note: open the bag containing the Coated Microplate only when it is at room temperature and close it immediately after use.

SAMPLE COLLECTION AND STORAGE
The assay should be performed using serum (standard sampling tubes or tubes containing serum separating gel) or plasma (lithium heparin, sodium heparin or potassium EDTA) samples.

QUALITY CONTROL
Good Laboratory Practice (GLP) requires the use of quality control specimens in each series of assays in order to check the performance of the assay. Controls should be treated as unknown samples, and the results analysed with appropriate statistical methods.
The kit control provided in the kit should be tested as unknown and is intended to assist in assessing the validity of results obtained with each assay plate.
The mean concentration of the control level is documented in the QC report included with each kit. These mean concentration levels are determined over several assays which are run in duplicate in multiple locations across each plate.
DiaMetra recommends the users to maintain graphic records of the control values generated with each assay run, including the running means, SDs and %CVs. This information will facilitate the controls trending analysis relating to the performance of current and historical control lots relative to the supplied Quality Control data. The trending will assist in the identification of assays which give control values significantly different from their average range.

CALCULATION OF RESULTS
A variety of data reduction software packages are available, which may be employed to generate the mean calibration curve and to calculate the mean concentrations of unknown samples and controls. A 4-parameter logistic (4PL) curve fit, including Calibrator 0 is required.
Alternatively, a calibration curve may be prepared on semilog graph paper by plotting mean absorbance on the Y-axis against concentration of analyte on the X-axis. Calibrator 0 should be included in the calibration curve. Read the mean absorbance value of each unknown sample off the curve.
In order for the assay results to be considered valid the kit calibrators and control must fall within the specifications detailed in the lot specific certificate of analysis.
If a control is out of its specified range, the associated test results are invalid and samples must be retested.