DiaMetra T4 Elisa Kit

PRINCIPLE 
The T4 (antigen) in the sample competes with the antigenic T4 conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti T4 coated on the microplate (solid phase) (the enzyme conjugate should have no measurable binding to serum proteins especially TBG and albumin).

CLINICAL SIGNIFICANCE
The thyroid hormone, thyroxine (T4) is produced by the thyroid gland. An important component in the synthesis is iodine. The major form of thyroid hormone in the blood is thyroxine (T4). Thyroxine is converted to the active T3 (three to four times more potent than T4) within cells by deiodinases (5'-iodinase).
Thyroxine-binding globulin (TGB) is the major carrier protein for circulating thyroid hormone. Only a very small fraction of the circulating hormone is free (unbound) - T4 0.03%.
The thyronines act on the body to increase the basal metabolic rate, affect protein synthesis and increase the body's sensitivity to catecholamines (such as adrenaline) by permissiveness. The thyroid hormones are essential to proper development and differentiation of all cells of the human body. These hormones also regulate protein, fat, and carbohydrate metabolism, affecting how human cells use energetic compounds. Numerous physiological and pathological stimuli influence thyroid hormone synthesis.
Thyroid hormones act through an unknown mechanism to inhibit neuronal activity; one of the effects is the reduction of the body temperature.
Thyrotoxicosis or hyperthyroidism is the clinical syndrome caused by an excess of circulating free thyroxine, free triiodothyronine, or both.
Both T3 and T4 are used to treat thyroid hormone deficiency (hypothyroidism).

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. The reagents are stable until the expiry date when stored and handled as 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, it 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.

QUALITY CONTROL
Each laboratory should assay controls at levels in the hypothyroid, euthyroid and hyperthyroid range for monitoring assay performance. These controls should be treated as unknowns and values determined in every test procedure performed. Quality control charts should be maintained to follow the performance of the supplied reagents. Pertinent statistical methods should be employed to ascertain trends. The individual laboratory should set acceptable assay performance limits. Other parameters that should be monitored include the 80, 50 and 20% intercepts of the calibration curve for run-to-run reproducibility. In addition, maximum absorbance should be consistent with past experience. Significant deviation from established performance can indicate unnoticed change in experimental conditions or degradation of kit reagents. Fresh reagents should be used to determine the reason for the variations.
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.

RESULTS
Mean Absorbance
Calculate the mean of the absorbance (Em) for each point of the calibration curve (C0-C5) and of each sample. 
Calibration curve
Plot the mean value of absorbance (Em) of the Calibrators (C0-C5) against concentration. Draw the best-fit curve through the plotted points. (es: Four Parameter Logistic).
Calculation of Results
Interpolate the values of the samples on the calibration curve to obtain the corresponding values of the concentrations expressed in μg/dL.