Endocrinology ELISA Assay

Thyroglobulin Ultrasensitive ELISA Kit

$650.00

The Thyroglobulin Ultrasensitive ELISA Assay Kit is an Enzyme-linked immunosorbent assay used for the quantitative and very sensitive determination of human thyroglobulin (hTg) in serum. The Eagle Biosciences Tg (Thyroglobulin) Ultrasensitive ELISA is for research use only and not for use in diagnostic procedures.

SKU: TGR31-K01 Categories: , ,

Thyroglobulin Ultrasensitive ELISA Kit

Thyroglobulin Ultrasensitive ELISA Kit Developed and Manufactured Medipan

Size: 1×96 wells
Sensitivity: 0.03 ng/mL
Dynamic Range: 0.03 – 3 ng/mL
Incubation Time: Overnight
Sample Type: Serum
Sample Size: 25 µL
Alternative Names: Tg Ultrasensitive ELISA, Human Thyroglobulin Ultrasensitive Assay, Human Tg Ultrasensitive ELISA, hTg Ultrasensitive ELISA
For Research Use Only

Assay Principle

The Thyroglobulin Ultrasensitive ELISA Kit is an immunoenzymometric assay (IEMA). One of the two specially selected monoclonal anti-Tg antibodies is immobilized onto the surface of microtiter plates. The other one is horseradish peroxidase (HRP) labeled and acts as conjugate. Both monoclonals are used in excess and bind Tg at different epitopes, which are relatively free from interferences.

Tg of serum samples, controls and calibrators react with the solid phase bound anti-hTg antibody during the first incubation period of 2.5 hours at room temperature. After removal of the non-bound components by a washing step anti-hTg HRP conjugate interacts with the Tg molecules trapped by the immobilized anti-hTg antibody during the second incubation at room temperature over night > 15 hours). The sandwich type com­plexes formed are separated from unbound material by a further washing step. HRP converts the colorless substrate solution of 3,3’,5,5’-tetramethylbenzidine (TMB) added into a blue product. The enzyme reaction is stopped by dispensing an acidic solution (H2SO4) into the wells after 15 min, turning the solution from blue to yellow.

The optical density (OD) of the solution at 450 nm is directly proportional to the amount of Tg bound. The standard curve is plotted by using the Tg concentrations of the calibrators (x-axis) and their corresponding OD values (y-axis) measured. The concentration of Tg of the specimen is directly read off from the standard curve.   If Tg values are above approx. 3 ng/ml, the sample has to be diluted 1 in 100 with the Tg-free serum diluent provided. These samples are again ana­lyzed. In that way, concentrations up to 300 ng Tg/ml become accessible. It is recommended to analyze the serum diluent G as unknown sample 1, representing the Tg-negative control. Its corresponding recovery sample GR serves as control for the 100 % value of the recovery experiment.

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Additional Information

Assay Background

Biochemically, Tg is to be understood as a rather complex family of molecules. It is micro heterogeneous with inter- and intra-individual variations (iodination degree, carbohydrate contents etc.). Dimers and several fragments also exist. Additional heterogeneity is due to malignant de-differentiation. Specifically and unspecifically interfering factors in individual sera cause further problems. Therefore, the Tg determination still represents a rather ambitious method.  On the other hand, Tg is the substratum of the thyroid hormone synthesis. Only thyroid tissue (even of malignant nature, if still differentiated) has the ability to produce, to store and to secrete Tg. Consequently, Tg is organ- and tissue specific.

Assay Procedure

  1. Bring all reagents to room temperature before use. Mix gently without causing foam.
  2. Dispense 25 µl mouse IgG (H) into the respective wells.
  3. Dispense 25 µl calibrators (alternatively plus calibrator 8), 25 µl controls (CI/II) diluted 1 in 10 or 1 in 100, 25 µl sample diluent (G) + 10 µl recovery sample (R), 25 µl neat patient serum, 25 µl corresponding serum + 10 µl recovery sample into the respective wells.
  4. Incubate 2.5 hours while shaking at room temperature (18 – 25 oC).
  5. Decant, then wash each well three times using 300 µl washing solution (prepared from B).
  6. Add 150 µl of conjugate solution (prepared from D and J) to each well.
  7. Incubate over night (> 15 h) at room temperature (18 – 25 oC).
  8. Decant, then wash each well three times using 300 µl washing solution.
  9. Add 100 µl of substrate (E) to each well.
  10. Incubate 15 min in the dark at room temperature (18 – 25 oC).
  11. Add 50 µl of stop solution (F) to each well and shake gently.
  12. Read the optical density at 450 nm versus 620 or 690 nm within 15 min after adding the stop solution.

Typical Standard Curve

Tg Standard Curve

Manual

Product Manual

Product Citations