ISO Thyroxine T4 Elisa Test Free T4 Serum Test 110 Minutes Read Time

T4 Thyroxine
REF: DS177703 96 tests

1. Intended use
Immunoassay for the in vitro quantitative determination of thyroxine in human serum.

2. Summary
The hormone thyroxine (T4) is the main product secreted by the thyroid gland and is an integral component of the hypothalamus-anterior pituitary-thyroid regulating system. It has the function of anabolically influencing metabolism. Thyroxine is formed in a coupling reaction from two DIT molecules (3,5-diiodotyrosine) in the thyroid gland. It is stored bound to thyroglobulin in the lumina of the thyroid follicles and is secreted as required under the influence of TSH. (1, 2)The major part (> 99 %) of total thyroxine (T4) in serum is present in proteinbound form. As the concentrations of the transport proteins in serum are subject to exogenous and endogenous effects, the status of the binding proteins must also be taken into account in the assessment of the thyroid hormone concentration in serum. If this is ignored, changes in the binding proteins (e.g. due to estrogencontaining preparations, during pregnancy or in the presence of a nephrotic syndrome etc.) can lead to erroneous assessments of the thyroid metabolic state. (3, 4, 5, 6, 7)The determination of T4 can be utilized for the following indications: the detection of hyperthyroidism, the detection of primary and secondary hypothyroidism, and the monitoring of TSH-suppression therapy. (8)
The T4 assay employs a competitive test principle with an antibody specifically directed against T4. Endogenous T4, released by the action of 8-anilino-1-naphthalene sulfonic acid (ANS).

3. Reagents Materials provided
• Coated Microplate, 8 x 12 strips, 96 wells, pre-coated with T4 derivant.
• Calibrators, 6 vials, 1 ml each, ready to use; Concentrations: 0(A), 2.5(B), 5(C), 10(D), 15(E) and 30(F) μg/dL.
• Enzyme Conjugate, 1 vial, 6.0 ml of HRP (horseradish peroxidase) labeled mouse monoclonal T4 in Tris-NaCl buffer containing BSA (bovine serum albumin). Contains 0.2% ProClin300 preservative. ANS 1.5 mg/mL.
• Substrate, 1 vial, 11ml, ready to use, (tetramethylbenzidine) TMB.
• Stop Solution, 1 vial, 6.0 ml of 1 mol/l sulfuric acid.
• Wash Solution Concentrate, 1 vial, 25 ml (40X concentrated), PBS-Tween wash solution.
• IFU, 1 copy.
• Plate Lid: 1 piece.

4. Materials required (but not provided)
• Microplate reader with 450nm and 620nm wavelength absorbent capability.
• Microplate washer.
• Incubator.
• Plate shaker.
• Micropipettes and multichannel micropipettes delivering 50μl with a precision of better than 1.5%.
• Absorbent paper.
• Distilled water

Test procedure

Ensure the patients’ samples, calibrators, and controls are at ambient temperature (18-25 ℃) before measurement. Mix all reagents through gently inverting prior to use.

• Use only the number of wells required and format the microplates’ wells for each calibrator and sample to be assayed. • Add 50 µL of calibrators or samples to each well.

• Add 50 µL of enzyme conjugate to each well.

• Shake the microplate gently for 30 seconds to mix.

• Cover the plate with a plate lid and incubate at 37 ℃ for 60 minutes.

• Discard the contents of the micro plate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper.

• Add 350 µl of wash solution, decant (tap and blot) or aspirate. Repeat 4 additional times for a total of 5 washes. An automated microplate strip washer can be used. At the end of washing, invert the plate and tap out any residual wash solution onto absorbent paper.

• Add 100 µL of substrate to each well.

• Cover and incubate at ambient temperature (18-25℃)in the dark for reaction for 20 minutes. Do not shake the plate after substate addition. • Add 50 µL of stop solution to each well.

• Shake for 15-20 seconds to mix the liquid within the wells. It is important to ensure that the blue color changes to yellow completely.

• Read the absorbance of each well at 450 nm ( using 620 to 630 nm as the reference wavelength to minimize well imperfections) in a micro plate reader. The results should be read within 30 minutes of adding the stop solution