DiaMetra Ferritin Elisa Kit

CLINICAL SIGNIFICANCE 
Ferritin is a protein mainly present in the cytoplasm but also found in serum and plasma as an iron-carrier protein. It contains, stores and releases iron in a controlled fashion. One molecule of ferritin is capable of binding between 4000 and 5000 atoms of iron, making ferritin the major iron storage protein for the body.
Iron is an essential micronutrient as it is required for an adequate erythropoietic function, oxidative mechanism and cellular immune response1,2,3. Under physiologic conditions there is a balance between iron absorption, iron transport and iron storage in the human body. When this balance is interrupted, two main conditions may present: iron deficiency and iron overload1,2,4. In humans, ferritin acts as a buffer against iron deficiency and overload.
Iron deficiency is the most common and widespread nutritional disorder in the world, affecting 2 billion people and in particular children, women of childbearing age with heavy menstrual flow and miscarriages, and premenopausal women. The WHO estimates that 30% of nonpregnant and 42% of pregnant women are affected by iron deficiency and anemia.
Since the concentration of ferritin is directly proportional to the total iron stores in the body, serum ferritin concentrations are considered as a common diagnostic tool in the diagnosis of diseases affecting iron metabolism.
In addition to ferritin levels additional markers assessed as part of the routine diagnosis of iron deficiency are transferrin saturation and the assessment of haemoglobin levels. All these parameters should also be considered together with other parameters such as the familial history of the patients and the evaluation of clinical symptoms.

PRINCIPLE OF THE METHOD
The Ferritin ELISA is based on simultaneous binding of human Ferritin to two monoclonal antibodies, one immobilised on the microwell plate and the other conjugated with horseradish peroxidase (HRP).
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 proportional to the ferritin concentration of in the sample.
Ferritin concentration in the sample is calculated through a calibration curve.

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 5 months. 
• The diluted wash solution is stable for 30 days at 2-8°C.

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.
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 cubic spline or 4-parameter logistic (4PL) curve fit, including Calibrator 0 is recommended.
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.