Hot Sale TSH ELISA TEST KIT
REF: DS177701 96 tests

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

1. Summary

• Thyroid-stimulating hormone (TSH, thyrotropin) is a glycoprotein having a molecular weight of approx. 30000 daltons and consisting of two subunits.
• Measurement of the serum concentration oftentimes thyrotropin (TSH), a glycoprotein with a molecular weight of 28,000 daltons and secreted from the anterior pituitary, is generally regarded as the most sensitive indicator available for the diagnosis of primary and secondary (pituitary) hypothyroidism (1,2).
• TSH measurements are equally useful in differentiating secondary and tertiary (hypothalamic) hypothyroidism from the primary thyroid disease. TSH release from the pituitary is regulated by thyrotropin releasing factor (TRH), which is secreted by the hypothalamus, and by direct action of T4 and triiodothyronine (T3), the thyroid hormones, at the pituitary.
• Increase levels of T3 and T4 reduces the response of the pituitary to the stimulatory effects of TRH. In secondary and tertiary hypothyroidism, concentrations of T4 are usually low and TSH levels are generally low or normal. Either pituitary TSH deficiency (secondary hypothyroidism) or insufficiency of stimulation of the pituitary by TRH (tertiary hypothyroidism) causes this.
• The TRH stimulation test differentiates these conditions. In secondary hypothyroidism, TSH response to TRH is blunted while a normal or delayed response is obtained in tertiary hypothyroidism.Further, the advent of immunoenzymometric assays has provided the laboratory with sufficient sensitivity to enable the differentiating of hyperthyroidism from euthyroid population and extending the usefulness of TSH measurements. This method is a second-generation assay, which provide the means for discrimination in the hyperthyroid-euthyroid range. (3)

2. Reagents Materials provided
• Coated Microplate, 8 x 12 strips, 96 wells, pre-coated with mouse monoclonal Anti-TSH.
• Calibrators, 6 vials, 1 ml each, ready to use; Concentrations: 0(A), 0.5(B), 2(C), 5(D), 10(E) and 25(F) μIU/mL.
• Enzyme Conjugate, 1 vial, 6.0 ml of HRP (horseradish peroxidase) labeled mouse monoclonal Anti-TSH in Tris-NaCl buffer containing BSA (bovine serum albumin). Contains 0.1% ProClin300 preservative.
• 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.

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

3. 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 25 µL of calibrators or samples to each well.

• Add 100 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