DiaMetra 17OH Progesterone Elisa Kit

CLINICAL SIGNIFICANCE
17-OH Progesterone (17-OH P) is an important intermediate of steroid biosynthesis. Elevated levels of 17-OH P are linked to dysfunction of steroid biosynthetic pathway that leads to an androgen excess. Therefore, measurements of 17-OH P are useful for the differential diagnosis of clinical conditions related to an hyperandrogenic phenotype and linked to adrenal disfunctions in both sexes.
Congenital Adrenal Hyperplasia (CAH) is a group of autosomal recessive disorders characterised by impaired cortisol synthesis1-4. The predominant cause of the condition (95% of cases) is mutations of the CYP21A2 gene that encodes the adrenal steroid 21-hydroxylase. 21- hydroxylase is responsible for conversion of 17-OHP to 11- deoxycortisol and progesterone to deoxycorticosterone. Assessment of 17-OH P levels provides supporting information for the differential diagnosis of CAH caused by mutations of the genes involved in cortisol synthesis from CYP21A2 gene mutations.
Measurement of 17-OH P is also considered useful in supporting diagnosis of other conditions characterised by hyperandrogenism such as polycystic ovarian syndrome (PCOS)5 and hirsutism in women, precocious puberty6 and precocious adrenarche. Determination of 17-OH P levels are used in such cases to exclude a diagnosis of CAH and non-classical CAH.

PRINCIPLE OF THE METHOD
The 17OH Progesterone ELISA is a competitive enzyme immunometric assay (ELISA) where 17OH Progesterone (antigen) in the sample competes with the antigenic 17OH Progesterone conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti 17OH 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 17OH Progesterone concentration of in the sample.
17OH 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 controls provided in the kit should be tested as unknowns and are intended to assist in assessing the validity of results obtained with each assay plate.
The mean concentration of each 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.

LIMITATIONS OF USE
As in the case of any diagnostic procedure, results must be interpreted in conjunction with the patient’s clinical presentation and other information available to the physician.
Heterophilic antibodies in human serum can react with reagent immunoglobulins, interfering with in vitro immunoassays9 . Patients routinely exposed to animals or to animal serum products can be prone to this interference and anomalous values may be observed.