DiaMetra T3 Elisa Kit

PRINCIPLE
The T3 (antigen) in the sample competes with the antigenic T3 conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti T3 coated on the microplate (solid phase) (the enzyme conjugate should have no measurable binding to serum proteins especially TBG and albumin).

CLINICAL SIGNIFICANCE
The triiodothyronine (T3), is a tyrosine-based hormones produced by the thyroid gland. An important component in the synthesis is iodine. Thyroxine-binding globulin (TGB) is the major carrier protein for circulating thyroid hormone. Only the free fraction of T3 is biologically active, a very small fraction of the circulating hormone is unbound - T3 0.3%.
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.
Both excess and deficiency of thyroxine can cause disorders. Thyrotoxicosis or hyperthyroidism is the clinical syndrome caused by an excess of circulating free thyroxine, free triiodothyronine, or both. It is a common disorder that affects approximately 2% of women and 0.2% of men. Hyperthyroidism is the case where there is a deficiency of thyroxine.
Release of T3 from the thyroid gland into the blood is ~5-8 micrograms/day. In addition, approximately 22 mg/day of T3 is produced by nonthyroidal 5- monodeionination of T4. T3 has a much faster turnover than T4 (T ~1 day, compared to ~6 days for T4) and has greater biological potency than T4.
Several drugs are known to affect the binding of Triiodothyronine to the thyroid hormone carrier proteins or its metabolism to T3 and complicate the interpretation of T3 results. Circulating autoantibodies to T3 and hormone-binding inhibitors may interfere
Measurement of total serum T3 concentrations is a standard and well-validated test of thyroid gland function.

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 tested. 
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 lipemeic or haemolysed should not be used in the assay.
Plate readers measure vertically. Do not touch the bottom of the wells.

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.

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. The conversion factor for the unit, sometimes reported in other systems is:
100 ng/dL = 1 ng/mL