CDSimple™ TSH Chemiluminescent ELISA Kit

Sample

Serum

Species Reactivity

Human

Intended Use

The Quantitative Determination of Thyrotropin Concentration in Human Serum by a Microplate Enzyme Immunoassay, Colorimetric.

Storage

1. Do not use reagents beyond the kit expiration date. 2. Avoid extended exposure to heat and light. Opened reagents are stable for sixty (60) days when stored at 2-8°C. Kit and component stability are identified on the label. 3. Above reagents are for a single 96-well microplate.

Performance Characteristics

Accuracy The TSH ELISA was compared with a reference immunochemiluminescence assay. Biological specimens from hypothyroid, euthyroid and hyperthyroid populations were used (The values ranged from 0.01μIU/ml – 61μIU/ml). The total number of such specimens was 241. The least square regression equation and the correlation coefficient were computed for the TSH ELISA method in comparison with the reference method. The data obtained is displayed in Table. Only slight amounts of bias between the TSH ELISA method and the reference method are indicated by the closeness of the mean values. The least square regression equation and correlation coefficient indicates excellent method agreement. Correlation between 1 hr and 2 hr incubation The one- (1) hr and two (2) hr (optional) incubation procedures were compared. Thirty (30) biological specimens (ranging from 0.1 – 18.5 μIU/ml) were used The least square regression equation and the correlation coefficient were computed for the 2 hr procedure (y) in comparison with the 1 hr method (x). Excellent agreement is evidenced by the correlation coefficient, slope and intercept: Y = 0.986 (x) + 0.119 Regression Correlation =0.998

Precision

The within and between assay precisions of the TSH ELISA were determined by analyses on three different levels of pool control sera. The number (N), mean (X) value, standard deviation (σ) and coefficient of variation (C.V.) for each of these control sera are presented in Tables

Sensitivity

The sensitivity (detection limit) was ascertained by determining the variability of the 0 μIU/ml serum calibrator and using the 2σ (95% certainty) statistic to calculate the minimum dose: For I hr incubation = 0.078 μIU/ml For 2 hr incubation = 0.027 μIU/ml

General Description

Measurement of the serum concentration of 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. The structure of human TSH is similar to that of the pituitary and placental gonadotropins, consisting of an 89-amino acid α-subunit which is similar or identical between these hormones and a 115-amino acid βsubunit, which apparently confers hormonal specificity. The production of the 2 subunits is separately regulated with apparent excess production of the α-subunit. The TSH molecule has a linear structure consisting of the protein core with carbohydrate side chains; the latter accounts for 16% of the molecular weight. 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 provides the means for discrimination in the hyperthyroid-euthyroid range. The functional sensitivity (<20% between assay CV) of the one-hour procedure is 0.195 μIU/ml while the two-hour procedure has a functional sensitivity of 0.095μIU/ml. In this method, TSH calibrator, patient specimen or control is first added to a streptavidin coated well. Biotinylated monoclonal and enzyme labeled antibodies are added and the reactants mixed. Reaction between the various TSH antibodies and native TSH forms a sandwich complex that binds with the streptavidin coated to the well. After the completion of the required incubation period, the antibody bound enzyme-thyrotropin conjugate is separated from the unbound enzyme-thyrotropin conjugate by aspiration or decantation. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce color. The employment of several serum references of known thyrotropin levels permits construction of a dose response curve of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with thyrotropin concentration.

Standard Curve

The data presented in table and figure are for illustration only and should not be used in lieu of a dose response curve prepared with each assay.