DiaMetra FT4 Elisa Kit

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%. When thyroid hormone is bound, it is not active, so the amount of FT3/FT4 is what is important. For this reason, measuring total thyroxine in the blood can be misleading. The concentration of free thyroid hormones in the blood is regulated by a negative feedback mechanism involving TSH. The binding of T4 by TBG plays a key role in this feedback mechanism and the most significant changes that occur in T4 binding capacity are the result of alterations in TBG. Changes in the circulating levels of TBG will result in a proportional increase or decrease in the concentration of total T4. However, measurement of serum FT4 is unaffected by changes in T4 protein binding levels and therefore correlates well with the functional thyroid state in most individuals. Factors responsible for discrepancies between serum total T4 levels and true thyroid states include TBG concentration, estrogenic hormones (pregnancy, oral contraceptives and estrogen) and drugs that bind to TBG preventing its binding to FT4 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. 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).

PRINCIPLE
The free T4 (FT4, 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).

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 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.

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 he 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. 
f 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 ng/dL.