DiaMetra TSH Elisa Kit

CLINICAL SIGNIFICANCE
Thyroid-stimulating hormone (TSH or thyrotropin) is a glycopolypeptide hormone synthesised and secreted by the anterior pituitary gland which regulates the endocrine function of the thyroid gland. TSH consists of two subunits - alpha and beta. The  subunit is identical to that of human chorionic gonadotropin (HCG), luteinising hormone (LH) and follicle-stimulating hormone (FSH). The β (beta) subunit is unique to TSH, and therefore determines its function.
TSH stimulates the thyroid gland to secrete the hormones thyroxine (T4) and triiodothyronine (T3).
TSH production is controlled by a Thyrotropin Releasing Hormone (TRH). TRH is manufactured in the hypothalamus and transported to the pituitary gland, where it increases TSH production and release. Somatostatin is also produced by the hypothalamus and has an opposite effect on the pituitary production of TSH, decreasing or inhibiting its release.

PRINCIPLE OF THE METHOD
The essential reagents required for an immunoenzymatic assay include high affinity and specificity antibodies (enzyme and immobilised) with different and distinct epitope recognition, in excess, and native antigen.
In this method, TSH calibrators, patient specimens and/or controls containing the native antigen are first added to streptavidin coated wells. Biotinylated monoclonal and enzyme labelled antibodies are added and the reactants mixed: these antibodies have high affinity and specificity and are detected against distinct and different epitopes of TSH. Reaction between the various TSH antibodies and native TSH occurs in the microwells without competition or steric hindrance forming a soluble sandwich complex.
The interaction is illustrated by the following equation.

REAGENT STORAGE AND STABILITY
Store the kit at 2 – 8°C in the dark.
The kit is stable at 2 – 8°C until the expiry date stated on the external kit label.
Once opened, the calibrators are stable at 2 – 8°C for 6 months.
The diluted wash solution is stable for 30 days at 2 – 8°C.

CALCULATION OF RESULTS
A variety of data reduction software packages are available, which may be employed to generate the mean calibration curve and to calculate the mean concentrations of unknown samples and controls. A 4-parameter logistic (4PL) curve fit, or cubic spline including Calibrator 0 is required. Other curve fitting algorithms are not recommended.
Alternatively, a calibration curve may be prepared on semilog graph paper by plotting mean absorbance on the Y-axis against concentration of analyte on the X-axis. Calibrator 0 should be included in the calibration curve. Read the mean absorbance value of each unknown sample off the curve.
In order for the assay results to be considered valid the kit calibrators and control must fall within the specifications detailed in the lot specific certificate of analysis.
If a control is out of its specified range, the associated test results are invalid and samples must be retested.
NOTE: If the absorbance of a sample exceeds that of C6, the sample should be diluted in C0 and re-analysed. Correct the result using an appropriate dilution factor.

QUALITY CONTROL
Good Laboratory Practice (GLP) requires the use of quality control specimens in each series of assays in order to check the performance of the assay. Controls should be treated as unknown samples, and the results analysed with appropriate statistical methods.
The kit control provided in the kit should be tested as unknown and is intended to assist in assessing the validity of results obtained with each assay plate.
The mean concentration of the control level is documented in the QC report included with each kit. These mean concentration levels are determined over several assays which are run in duplicate in multiple locations across each plate.
DiaMetra recommends the users to maintain graphic records of the control values generated with each assay run, including the running means, SDs and %CVs. This information will facilitate the controls trending analysis relating to the performance of current and historical control lots relative to the supplied Quality Control data. The trending will assist in the identification of assays which give control values significantly different from their average range.