DiaMetra Aldosterone Elisa Kit

PRINCIPLE OF THE METHOD
The Aldosterone ELISA is a competitive enzyme immunometric assay (ELISA) where aldosterone (antigen) in the sample competes with the antigenic aldosterone conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti- aldosterone 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 aldosterone concentration in the sample.

Aldosterone concentration in the sample is calculated through a calibration curve.

CLINICAL SIGNIFICANCE
Aldosterone is a steroid hormone produced by the zona glomerulosa of the adrenal cortex in the adrenal gland. It plays a primary role in the regulation of sodium and potassium balance in the blood1 . Aldosterone secretion is stimulated primarily via the renin angiotensin-aldosterone system (RAAS). The production of renin is stimulated when there is a reduction in renal perfusion pressure, reduction in plasma volume and a negative sodium balance. Renin cleaves Angiotensinogen into Angiotensin I which ultimately leads to the production of Angiotensin II via angiotensin converting enzyme2 . Angiotensin II acts on the vascular system causing vasoconstriction as well as stimulating the adrenal cortex to secrete aldosterone. Aldosterone acts on mineralocorticoid receptors within the principal cells of the distal tubule to increase reabsorption of sodium and chloride and decrease reabsorption of potassium and hydrogen. Aldosterone measurements are used in the diagnosis and treatment of primary aldosteronism3,4 (a disorder caused by excessive secretion of aldosterone by the adrenal gland), hypertension caused by primary aldosteronism, selective hyperaldosteronism, edematous states and other conditions of electrolyte balance.

PRECAUTIONS
Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance data represented here were obtained using specific reagents listed in this Instruction For Use.
All reagents should be stored refrigerated at 2-8°C in their original container. Any exceptions are clearly indicated.
Allow all kit components and specimens to reach room temperature (22-28°C) and mix well prior to use.
Do not interchange kit components from different lots. The expiry date printed on box and vials labels must be observed. Do not use any kit component beyond their expiry date.
If you use automated equipment, the user has the responsibility to make sure that the kit has been appropriately validated for its intended use/purpose.
The incomplete or inaccurate liquid removal from the wells could influence the assay precision and/or increase the background. To improve the performance of the kit on automatic systems is recommended to increase the number of washes.
It is important that the time of reaction in each well is held constant for reproducible results. Pipetting of samples should not extend beyond ten minutes to avoid assay drift. If more than 10 minutes are needed, follow the same order of dispensation. If more than one plate is used, it is recommended to repeat the dose response curve in each plate
Addition of the TMB Substrate solution initiates a kinetic reaction, which is terminated by the addition of the Stop Solution. Therefore, the TMB Substrate and the Stop Solution should be added in the same sequence to eliminate any time deviation during the reaction.
Observe the guidelines for performing quality control in medical laboratories by assaying controls and/or pooled sera.
Maximum precision is required for reconstitution and dispensation of the reagents. 
Samples microbiologically contaminated, highly lipemic, icteric or haemolysed should not be used in the assay.
Plate readers measure vertically. Do not touch the bottom of the wells.
Fresh disposable tips must be used when pipetting assay reagents including samples, calibrators and controls to mitigate the risk of carryover contamination. Failure to do so may lead to invalid results.

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. 
in use stability data supports reagent stability when used two times within this period.
Important note: open the bag containing the Coated Microplate only when it is at room temperature and close it immediately after use.

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 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.
When interpreting control data, users should note that this product was designed and developed as a manual product. The range stated on the QC certificate should be appropriate for assays that are performed manually and with strict adherence to the Assay Procedure described above. It is recognised by Quality Control professionals, that as a result of differences in conditions and practices, there will always be variability in the mean values and precision of control measurements between different laboratories.

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.
The performance characteristics of this assay have not been established in a paediatric population.
Heterophilic antibodies in human serum can react with reagent immunoglobulins, interfering with in vitro immunoassays. Patients routinely exposed to animals or to animal serum products can be prone to this interference and anomalous values may be observed.