Anti-Tg (Thyroglobulin) ELISA

$340.00

The Eagle Biosciences Anti-Tg (Thyroglobulin) ELISA Assay kit is intended for the quantitative determination of Anti-Tg (Thyroglobulin) in serum by enzyme linked immunoassay (ELISA). The Anti-Tg ELISA Assay kit is for research use only and not to be used in diagnostic procedures.

SKU: ATG31-K01 Categories: , ,

Anti-Tg (Thyroglobulin) ELISA

For Research Use Only

Size: 1×96 wells
Sensitivity: 1 U/mL
Dynamic Range: 20 – 1280 U/ml
Incubation Time: 2 hours
Sample Type: Serum
Sample Size: 10 µL

Controls Included

Product Developed and Manufactured in the USA

Additional Information

Assay Background

It is a routine practice of measuring serum autoantibodies to thyroglobulin (Tg) and microsomal (TPO) for aid in detecting and monitoring autoimmune thyroid disease. Serum anti-TPO autoantibody and anti-Tg autoantibody are found to be well- correlating with histological changes in Harshimoto’s thyroiditis. Clinically, positive anti-TPO autoantibody is detected in patients with chronic thyroiditis (70-90%), primary hypothyroidism (~60%), thyrotoxicosis (~50%) and thyroid tumors (~17%), however, anti-Tg autoantibody is mainly identified in patients with Harshimoto’s thyroiditis and Graves’ disease (40-70%).

Although ELISA technology has applied to detecting these autoantibodies, the high background in normal population would decrease the clinical diagnostic sensitivity and specificity. This high sensitive anti-Tg autoantibody ELISA kit was developed with proprietary technology that leads to a very low reaction background in normal population and thus would increase the clinical diagnostic sensitivity and specificity.

Assay Principle

The Eagle Biosciences anti-Tg autoantibody IgG ELISA Assay Kit is designed, developed and produced for the quantitative measurement of human anti-Tg IgG autoantibody level in test sample. Assay calibrators, controls and pre-diluted human serum samples containing anti-Tg IgG autoantibody are added to microtiter wells of microplate that was coated with high affinity streptavidin on its wall. The autoantibody reaction will not start until the addition of a biotinylated human Tg antigen. After the first incubation period, the unbound protein matrix is removed in the subsequent washing step. A horseradish peroxidase-conjugated rabbit anti-human IgG subclass specific antibody (tracer antibody) is added to each well.

After an incubation period an immunocomplex of “solid-phase bound biotin-Tg – human anti-Tg IgG – HRP-conjugated tracer antibody” is formed if there is human anti-Tg IgG autoantibody present in the test sample. The unbound tracer antibody is removed in the subsequent washing step. HRP-conjugated tracer antibody bound to the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the tracer antibody bound to the human IgG on the wall of the microtiter well is directly proportional to the amount of human anti-Tg IgG autoantibody level in the sample. Plotting the absorbance versus the respective human anti-Tg IgG autoantibody concentration for each calibrator on point-to-point or 4-parameter fit generates a calibrator curve. The concentration of human anti-Tg IgG autoantibody in test samples is determined directly from this calibrator curve.

Assay Procedure

  1. Add 25 µL of calibrators, controls and diluted patient serum samples into the designated microwell.
  2. Add 100 µL of biotinylated Tg solution into each well.
  3. Cover the plate with one plate sealer.
  4. Incubate plate at room temperature for 1 hour.
  5. Prepare Anti-hIgG Tracer Antibody Working Solution by 1:21 fold dilution of the tracer antibody with the Tracer Antibody Diluent. For each strip, it is required to mix 1 mL of Tracer Antibody Diluent with 50 µL of the Tracer Antibody in a clean test tube.
  6. Remove the plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL to 400 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
  7. Add 100 µL of above diluted tracer antibody working solution to each of the wells.
  8. Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
  9. Incubate plate at room temperature for 30 minutes.
  10. Remove the plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL to 400 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
  11. Add 100 µL of ELISA HRP Substrate into each of the wells.
  12. Cover the plate with a new plate sealer and also with aluminum foil to avoid exposure to light.
  13. Incubate plate at room temperature for 20 minutes.
  14. Remove the aluminum foil and plate sealer. Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
  15. Read the absorbance at 450 nm within 10 minutes in a microplate reader.

Typical Standard Curve

Anti-Tg (Thyroglobulin) ELISA

Manual

Product Manual