Thyroxine T4 Elisa Diagnostic Kits Plasma Sample With 2 Standard Curve Ranges

Thyroxine T4 ELISA test , high accuracy, Elisa Sandwich method, for quantitative measurement

Product Name: Thyroxine T4 ELISA test

Intended Use:

Thyroxine (T4) kit is designed to quantitatively measure T4 present in serum, plasma (EDTA and Heparin), urine, extracted dried fecal samples, and tissue culturemedia. This kit measures totalT4 in serum, plasma, and extracted fecal samples. T4 is species independent.

Summary:

Thyroxine is the main hormone produced by the thyroid gland. The thyroid hormones, triiodothyronine (T3) and thyroxine (T4), are tyrosine-based hormones produced by the thyroid gland that are primarily responsible for regulation of metabolism. Iodine is necessary for the production of T3 and T4. A deficiency of iodine leads to decreased production of T3 and T4, enlarges the thyroid tissue and will cause the disease known as goitre. The major form of thyroid hormone in the blood is thyroxine (T4), which has a longer half-life than T3. The ratio of T4 to T3 released into the blood is roughly 20 to 1. T4 is converted to the active T3 (three to four times more potent than T4) within cells by deiodinases (5’-iodinase). These are further processed by decarboxylation and deiodination to produce iodothyronamine (T1a) and thyronamine (T0a). All three isoforms of the deiodinases are selenium-containing enzymes, thus dietary selenium is essential for T3 production. Hypothyroidism is the condition that results from under-production of thyroxine by the thyroid gland either because the gland is naturally underactive or because radioiodine therapy or surgery for an overactive gland has resulted in underactivity. Thyroxine is taken to replace the deficiency which exists in such situations and therefore to restore normal metabolic activity. Thyroid hormone production is regulated via pituitary thyrotropin (TSH) modulation of thyroxine (T4) prohormone secretion by the thyroid gland and regulation of active triiodothyronine (T3) production in peripheral tissues via metabolic events influencing enzyme.

Principle of Test:

The kit offers 2 standard curve ranges. For serum and plasma samples, we recommend using 10 µL of standards or samples. The assay concentration range for T4 will be from 50 ng/ml to 0.781 ng/ml. For urine samples, we recommend alternatively using 100 µL of standards or samples concentrations of T4 that range from 4 ng/ml to 0.0625 ng/ml.

A T4 stock solution is provided to generate standard curves for the assay and all samples should be read off the standard curve. Standards or diluted samples are pipetted into a clear microtiter plate coated with an antibody to capture mouse antibodies. A T4-peroxidase conjugate is added to the standards and samples in the wells. The binding reaction is initiated by the addition of a monoclonal antibody toT4 to each well. After an hour incubation the plate is washed and substrate is added. The substrate reacts with the bound T4-peroxidase conjugate. After a short incubation, the reaction is stopped and the intensity of the generated color is detected in a microtiter plate reader capable of measuring 450nm wavelength. The concentration of the T4 in the sample is calculated, after making suitable correction for the dilution of the sample, using software available with most plate readers.

Kit Components

1.  Microtiterwells, 12 x 8 (break apart) strips, 96 wells; Wells coated with Treponema pallidum antigen. (incl. 1 strip holder and 1 cover foil)
2.  Pos. Control ***, 1 vial, 1.0 mL, ready to use; colored yellow, red cap.
3.  Neg. Control ***, 1 vial, 2.0 mL, ready to use; colored yellow, yellow cap.
4.  Cut-off Control ***, 1 vial, 1.0 mL, ready to use; colored yellow, black cap.
5.  Enzyme Conjugate **, 1 vial, 13 mL, ready to use,
6.  Substrate Solution, 1 vial, 14 mL, ready to use, Tetramethylbenzidine (TMB).
7.  Stop Solution, 1 vial, 14 mL, ready to use, contains 0.2 mol/l H2SO4, Avoid contact with the stop solution. It may cause skin irritations and burns.
8.  Wash Solution *, 1 vial, 30 mL (20X concentrated for 600 mL), pH 7.2 ± 0.2 see „Preparation of Reagents“.

* contains 0.03 % ProClin 300

** contains 0.03 % ProClin 300 + 0.01 % Gentamicin sulphate

*** contains 0.03 % ProClin 300 + 0.015 % 5-bromo-5-nitro-1,3-dioxane (BND) + 0.010 % 2-methyl-2H-isothiazol-3-one (MIT)

ASSAY PROCEDURE

Each run must include a standard curve.

1. Secure the desired number of Microtiter wells in the frame holder.

2. Dispense 10 µL of each Standard, Control and samples with new disposable tips into appropriate wells.

3. Incubate for 5 minutes at room temperature (18 °C – 25 °C).

4. Dispense 100 µL Enzyme Conjugate into each well.

Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

5. Incubate for 80 minutes at room temperature (18 °C – 25 °C).

6. Briskly shake out the contents of the wells.

Rinse the wells 5 times with diluted Wash Solution (400 µL per well). Strike the wells sharply on absorbent paper to remove residual droplets.

Important note:

The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing procedure!

       1. Add 100 µL of Substrate Solution to each well.

       2. Incubate for 10 minutes at room temperature (18 °C – 25 °C) Incubate for 7 minutes at room      temperature (26 °C – 29 °C) Incubate for 5 minutes at room temperature (more than 29 °C)

       3. Stop the enzymatic reaction by adding 100 µL of Stop Solution to each well.

        4. Determine the absorbance (OD) of each well at 450 ± 10 nm with a microtiter plate reader.

            It is recommended that the wells be read within 10 minutes after adding the Stop Solution.