The PRIMATE TSH ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system utilizes affinity purified antibody directed against intact PRIMATE TSH molecule for solid phase (microtiter wells) immobilization and a mouse anti bovine TSH beta antibody is in the antibody-enzyme (horseradish peroxidase) conjugate. The test sample is allowed to react simultaneously with the two antibodies, resulting in the TSH molecules being sandwiched between the solid phase and enzyme-linked antibodies. After a 1 hours of incubation period at 37 C, the wells are washed with wash solution to remove unbound-labeled antibodies. A solution of TMB is added and incubated for 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of stop solution and the color is changed to yellow and measured spectrophotometrically at 450 nm. The concentration of TSH is directly proportional to the color intensity of the test sample.
< Examples of TSH Standard Curve >
A typical standard curve (illustration only) for PRIMATE TSH is given below:
|OD at 450nm
<ELISA Performance Characters>
Precision: Inter and Intra assay variation (CV) were determined from different pooled serum samples in threedifferent experiments.
|Inter-assay variation||Set1: CV= 5.9% (N=10)||Set2: CV= 6.4 % (N=10)||Set3: CV= 4.4 % (N=10)|
|Intra-assay variation||Set1: CV= 8.9% (N=10)||Set2: CV= 5.4 % (N=10)||Set3: CV= 8.4 % (N=10)|
< Sensitivity >
The lowest level detectable in this assay is 0.5ng/mL of serum or plasma
< Specificity >
The PRIMATE TSH ELISA system utilizes monoclonal antibody and high affinity polyclonal antibody to TSH. The cross reactivity to other pituitary gonadotropins (PRIMATE LH, FSH) is not detectable under the conditions of the assay system.
1. Antibody-coated microtiter wells
2. Reference standard, Ready to use (0, 1, 2.5, 5, 10, 25, ng/mL), 0.6mL/vial
3. Enzyme Conjugate Reagent, 12 mL
4. TMB color reagent (ready to use) , 12 mL
5. 20X Wash buffer, 20 mL
6. Stop solution (2N HCl), 6mL
7. Sample diluent, 20ml
Thyroid Stimulating Hormone (TSH) ELISA