Free T4 (Thyroxin) MICRO-ELISA Test Kit

COMPONENTS IN EACH 96 TEST MICRO-EIA fT4 DIAGNOSTIC KIT
1. 96 wells T₄ ANTIBODY COATED WELLS Coated with anti T₄ (sheep polyclonal); contained in a pack with silica gel desiccant.
2. 1 bottle 10.5 mL T₄ ENZYME CONJUGATE T₄-labeled horseradish peroxidase in a buffered protein solution; contains a preservative.
3. 1 vial 0.75 mL Free T₄ SERUM STANDARD, 0 ng/dl. Human serum; contains a preservative.
4. 5 vials 0.75 mL Free T₄ SERUM STANDARDS, 0.4, 1.10, 2.00, 3.80 AND 7.50 ng/dl. Human serum with added T₄; contains a preservative.
NOTE: Exact value of Standards are lot specific and are listed on the vial label.
5. 1 bottle 50.0 mL WASH BUFFER CONCENTRATE (20X) Buffered detergent solution; contains a preservative. Dilute bottle to 1000 mL with deionized water.
6. 1 bottle 12.0 mL SUBSTRATE CHROMOGEN Buffered hydrogen peroxide and 3,3',5,5' tetramethylbenzidine (TMB) solution.
7. 1 bottle 12.0 mL STOP SOLUTION 1 N H ₂SO₄.

ADDITIONAL REQUIRED MATERIALS

Disposable tip precision pipettes 0.050, and 0.1 mL
microtiter plate reader
Distilled or deionized water

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Patient sample may contain pathogens: treat all samples as potentially
infectious.

CAUTION: Source material used to prepare Standards was derived from human material. The material was tested using FDA-approved methods and found non-reactive for Hepatitis B Surface Antigen (HBsAg) by ELISA and non-reactive for HIV by ELISA. No known test method can offer total assurance that infectious agents are absent.

HANDLE THESE REAGENTS AS IF THEY ARE POTENTIALLY INFECTIOUS.
Information on handling human serum is provided in the CDC/NIH manual "Bio-safety in Microbiological and Biomedical Laboratories" (1984).

Good laboratory practice requires that quality control specimens be run with each patient sample run to check the assay performance. Three controls with normal, low and elevated values should be used. Pooled human serum or commercially available control sera are suitable. Any material used should be assayed repeatedly to establish mean values and acceptable ranges to assure proper performance.

Do not mix or interchange reagent from kits with different lot numbers. Pool and mix reagents from different bottles before use.

Do not use reagents beyond the expiration date printed on each vial or bottle.

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1. Pipet 50.0 µL of Free T₄ standards into the appropriate well. (Only the 0 ng/dl standard need be run if
using the previously stored curve).
2. Pipet 50.0 µL of each control and patient serum into the appropriate well.
3. Pipet 100 µL (0.1 mL) of Free T₄-enzyme conjugate into all wells and mix gently.
4. Incubate at room temperature (18°- 30°C) for 60 minutes ± 5 minutes.
5. Decant or aspirate and discard liquid contents of all wells.
6. Fill each well with diluted WASH BUFFER. Decant or aspirate liquid contents of all wells. Do not use tap water.
WARNING: WASHING THE WELLS IS OF CRITICAL IMPORTANCE. Fill the wells to overflowing, you CANNOT cause any carryover between wells. You CANNOT over wash the wells. Completely decant or aspirate all of the liquid out of the wells. SLAP the inverted wells on a fresh clean piece of absorbent paper AFTER EVERY WASH.
YOU CANNOT SLAP TOO HARD, REMOVE ALL OF THE LIQUID FROM THE WELLS.
7. Repeat step 6 twice more (for a total of 3 washes). Tap wells free of any liquid or aspirate thoroughly.
8. Pipet or dispense 100 µL (0.1 mL) of SUBSTRATE / CHROMOGEN REAGENT into each well.
9. Mix thoroughly and incubate 15 minutes at room temperature (18°-30°C).
10. Pipet or dispense 100 µL (0.1 mL) of STOPPING REAGENT into each well and mix thoroughly.
11. Read the absorbance of each well at 450 ± 20 nm against water.

1. Calculate the %A/A₀ value for each standard, control and sample.

		A
%A/A0	=		×	100%
		A0
A = the average absorbance value for the standard, controlsand patient samples.
A0 = the average absorbance value for the 0 ng/dl standard.

2. Construct a standard curve by plotting the %A/A₀ value for the thyroxin
standards (vertical axis) versus the T₄ standard concentration (horizontal
axis) on the logit-log graph paper supplied
3. Draw the best straight line through the points.
4. Interpolate the control and patient sample values from each %A/A₀
value obtained.
5. Save the calibration curve for use in subsequent runs using only single point calibration (0 ng/dl). Optional calculation method for use with single point calibration and stored standard calibration curve.

a. For single point calibration runs, calculate %A/A₀ values for samples
and controls using the absorbance of the 0 ng/dl standard that was run
with them.
b. Using the original standard calibration curve, interpolate the control
and patient sample values from each %A/A₀ value obtained in the run.
NOTE: For automated data reduction, use a log/logit date transformation
of A/A₀ vs. T₄ concentration.

 

EXAMPLE DATA

Specimen I.D		A450	%A/A0	Calculated Value
STANDARD	0 ng/dl	3.642 4.344	100%
STANDARD	0.35 ng/dl	3.425 3.463	86%
STANDARD	1.00 ng/dl	2.844 2.867	72%
STANDARD	2.10 ng/dl	1.925 2.009	49%
STANDARD	3.30 ng/dl	0.873 0.863	22%
STANDARD	7.50 ng/dl	0.320 0.374	10%
PATIENT	1	3.298 3.274	82%	0.58 ng/dl
PATIENT	2	2.017 2.076	51%	1.64 ng/dl
PATIENT	3	1.034 1.006	26%	3.69 ng/dl

The range of this assay is 0 - 7.6 ng/dl. For specimen with T₄concentrations beyond the standard curve (7.6 ng/dl), repeat the test by diluting the specimen with the 0 ng/dl Standard. To obtain the final concentration, multiply the concentration of the diluted sample by the dilution factor.

The principal tests used in the laboratory evaluation of thyroid function are Total Thyroxin (T₄), Total Triiodothyronine (T₃), T-Uptake (T-Up), a calculated Free Thyroxin Index (FTI) and Thyroid Stimulating Hormone (TSH). The results of these tests are interrelated and help the clinician in making a diagnosis. Clinical hypothyroidism results from underproduction of thyroid hormones by the thyroid gland, consequently an abnormally low circulating T₄ and T₃ concentration in blood. Clinical hyperthyroidism results from excessive production of thyroid hormones and resulting elevation of T₄ and T₃ concentrations. The manifestations of thyroid dysfunction can result from disease of the thyroid gland (PRIMARY hyperthyroidism or hypothyroidism), disease of the pituitary gland (SECONDARY hyperthyroidism or hypothyroidism) or disease of the hypothalamus (TERTIARY hyperthyroidism or hypothyroidism).
Thyroxin (3,5,3',5'-tetraiodo-l-thyronine, T₄) and Triiodothyronine (3,5,3'- triiodo-l-thyronine, T₃), are the hormones originating from the thyroid gland. T₄ and T₃ are responsible for regulating diverse biochemical processes throughout the body that are essential for protein synthesis, normal development, metabolic and neural activity. T₄ is synthesized within the thyroid gland and secreted directly into the bloodstream. Approximately 30% of the circulating T₄ is enzymatically deiodinated at the 5' position in the peripheral tissues to yield T₃. The T₄ likely serves as a "prohormone" for T₃, which has a much greater metabolic activity.
T₄ and T₃ are transported through the peripheral blood stream largely bound to serum proteins. The major transport protein is Thyroxin Binding Globulin (TBG) which normally accounts for 80% of the bound hormone. The other thyroid hormone binding proteins are Thyroxin Binding Prealbumin and Albumin. Only about 0.3% of the total serum T₃ and only about 0.1% of the total serum T₄ are unbound and free to diffuse into tissue to exert their biological effects. When the level of TBG increases, the level of total T₄ will increase to maintain the same level of unbound or free T₄ in the bloodstream of an euthyroid individual. Simply determining the total T₄ concentration fails to take into account the variations in TBG levels that affect the unbound thyroxin (free T₄) concentration. TBG levels can vary for reasons incidental to the patient's thyroid status such as the presence of certain drugs, steroid hormones, pregnancy, and various non-thyroidal diseases. The Thyroid Uptake (TUp) test is an indirect measurement of empty binding sites for T₄ on the TBG molecule (unsaturated TBG) in the patient specimen. The number obtained from the multiplication of the Total T₄ concentration by the Thyroid Uptake value is called a Free Thyroxin Index (FTI). The FTI correlates more closely with Free T₄ (the metabolic active fraction) concentration than does the total T₄ concentration alone. The FTI is therefore a better method of monitoring thyroid function and diagnosing thyroid illness than is a Total T₄ determination alone. Diseases of the thyroid gland can result in clinical signs of thyroid dysfunction. Primary hypothyroidism results in underproduction of T₄ by the thyroid gland and consequently an abnormally low circulating T₄ concentration in the blood. Primary hyperthyroidism leads to excessive thyroid production of T₄ and a resulting elevated T₄ concentration. The determination of total serum T₃ is used in the differential diagnosis of thyroid disease, particularly hyperthyroidism. In most hyperthyroid patients, both serum T₃ and T₄ are elevated. However, approximately 5- 10% of hyperthyroid patients have elevated T₃ concentrations but normal serum T₄, a condition known as T₃-thyrotoxicosis. Such clinical conditions make it vital to establish that serum T₃ is normal before excluding the diagnosis of hyperthyroidism. Serum T₃ level is also an excellent indicator for the ability of the thyroid to respond to both stimulatory and suppressive tests.
The thyroid gland function is regulated by the level of Thyroid Stimulating Hormone (TSH) which is produced and secreted by the pituitary gland. TSH is produced by the anterior lobe of the pituitary gland and acts on the thyroid gland to release thyroid hormones. The release of TSH from the pituitary is regulated by the hypothalamus when it secretes TRH (thyrotropin releasing hormone).
In an euthyroid individual, the levels of thyroid hormones in the blood are inversely related to the levels of TSH and TRH. When the levels of thyroid hormones rise, the levels of TRH and TSH fall; and when the levels of thyroid hormones fall, the levels of TRH and TSH rise. In the event of failure of the thyroid gland, the levels of thyroid hormones fall and the negative feedback results in an elevated level of TSH in the blood. Elevated levels of TSH are thus useful in the diagnosis of primary hypothyroidism. Conversely, in the case of primary hyperthyroidism, the elevated levels of thyroid hormones will result in decreased levels of TSH. When there is a failure of the pituitary or the hypothalamus (secondary or tertiary hypothyroidism), the level of TSH is decreased in the presence of low levels of thyroid hormones. In secondary or tertiary hyperthyroidism, the level of TSH is increased in the presence of high levels of thyroid hormones.

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