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
