Anti Thyroid Peroxidase Elisa Test Kit Anti TPO Antibody Test Kits

Anti-TPO Anti-Thyroid Peroxidase Elisa Kit

1. Intended use

Immunoassay for the in vitro quantitative determination of antibodies to thyroid peroxidase in human serum. The anti‐TPO determination is used as an aid in the diagnosis of autoimmune thyroid diseases.

2. Summary 

• Thyroid‐specific peroxidase (TPO) is present on the microsomes of thyrocytes and is expressed at its apical cell surface. In synergy with thyroglobulin (Tg) this enzyme has an essential function in the iodination of L‐tyrosine and the chemical coupling of the resulting mono‐ and di‐iodotyrosine to form the thyroid hormones T4, T3, and fT3.
• TPO is a potential autoantigen. Elevated serum titers of antibodies to TPO are found in several forms of thyroiditis caused by autoimmunity. The still frequently found term “microsomal antibody” originates from the time when TPO had not yet been identified as an antigen in autoimmunity caused by microsomes. In the clinical sense the two terms anti‐ TPO and microsomal antibody can be used synonymously; there are differences, however, with regard to the test methods.
• High anti‐TPO titers are found in up to 90 % of patients with chronic Hashimoto's thyroiditis. In Graves' disease, 70 % of the patients have an elevated titer. Although the sensitivity of the procedure can be increased by simultaneously determining other thyroid antibodies (anti‐Tg, TSH‐receptor‐antibody - TRAb), a negative finding does not rule out the possibility of an autoimmune disease. The magnitude of the antibody titer does not correlate with the clinical activity of the disease. Initially elevated titers can become negative after lengthy periods of illness or during remission. If antibodies reappear following remission, then a relapse is probable.
• Whereas the usual microsomal antibody tests employ unpurified microsomes as an antigen preparation, the anti‐TPO tests use a purified peroxidase. The two procedures are of comparable performance in terms of clinical sensitivity, but better lot‐to‐lot consistency and higher clinical specificity can be expected from anti‐TPO tests due to the higher quality of the antigen used. The Enzyme linked immunosorbent assays uses human antigen and rabbit anti-human IgG antibodies (anti-IgG).

3. Test principle

Indirect method, total duration of assay: 70 minutes.

The Anti-TPO ELISA employs solid phase, indirect ELISA method for detection of antibodies to TPO in two-step incubation procedure. Polystyrene microwell strips are pre- coated with highly immunoreactive human TPO antigens. During the first incubation step, anti-TPO specific antibodies, if present, will be bound to the solid phase pre-coated TPO antigens. The wells are washed to remove unbound serum proteins, and rabbit anti-human IgG antibodies (anti-IgG) conjugated to the enzyme horseradish peroxidase (HRP- Conjugate) are added. During the second incubation step, these HRP-conjugated antibodies will be bound to any antigen-antibody(IgG) complexes previously formed and the unbound HRP-conjugate is then removed by washing. Chromogen solutions containing Tetramethylbenzidine (TMB) and urea peroxide are added to the wells and in presence of the antigen-antibody-anti-IgG (HRP) immunocomplex, the colorless Chromogens are hydrolyzed by the bound HRP conjugate to a blue-colored product. The blue color turns yellow after stopping the reaction with sulfuric acid.

The amount of color intensity can be measured and it is proportional to the amount of antibody captured in the wells, and to the amount of antibody in the sample respectively.

4. Reagents Materials provided

• Coated Microplate, 8 x 12 strips, 96 wells. Pre-coated with human TPO antigen.

• Calibrators, 6 vials, 1 mL each, ready to use; Concentrations: 0(A), 25(B) ,50(C), 100(D), 250(E) and 500(F) IU/mL.

• Enzyme Conjugate, 1 vial, 11.0 mL of HRP (horseradish peroxidase) labeled rabbit anti- human IgG antibodies (anti-IgG) in Tris-NaCl buffer containing BSA (bovine serum albumin). Contains 0.1% ProClin300 preservative.

• Serum Diluent: 1 vial, 11mL. Containing buffer salts and a dye

• Wash Solution Concentrate, 1 vial, 25 ml (40X concentrated), PBS-Tween wash solution.

• Substrate, 1 vial, 11ml, ready to use, (tetramethylbenzidine) TMB.

• Stop Solution, 1 vial, 6.0 ml of 1 mol/l sulfuric acid.

• IFU, 1 copy.

• Plate Lid: 2 pieces.

Materials required (but not provided)

• Microplate reader with 450nm and 620nm wavelength absorbent capability.

• Microplate washer.

• Incubator.

• Plate shaker.

• Micropipettes and multichannel micropipettes delivering 50µl with a precision of better than 1.5%.

• Absorbent paper.

• Distilled water

5. Test procedure

• Use only the number of wells required and format the microplate wells for each calibrator and sample to be assayed.

• Add 100 µL of calibrators to each well.

• Add 100 µL of Sample Diluent (Green color) to each well Except the Calibrator-wells.

• Add 10 µL of Sample to each Sample Diluent well (NOTE: Reagents in Wells will turn Blue color from Green), then shake 30 seconds.

• Cover the plate with a plate lid and incubate at 37 °C for 30 minutes.

• Discard the contents of the micro plate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper.

• Add 350 µL of wash solution, decant (tap and blot) or aspirate. Repeat 4 additional times for a total of 5 washes. At the end of washing, invert the plate and tap out any residual wash solution onto absorbent paper.

• Add 100 µL of enzyme conjugate,

• Cover the plate with a plate lid and incubate at 37 °C for 30 minutes.

• Add 350 µL of wash solution, decant (tap and blot) or aspirate. Repeat 4 additional times for a total of 5 washes. At the end of washing, invert the plate and tap out any residual wash solution onto absorbent paper.

• Add 100 µL of Substrate to each well. Cover and incubate at ambient temperature (18-25℃) in the dark for reaction for 10 minutes. Do not shake the plate after substate addition.

• Add 50 µL of stop solution to each well.

• Shake for 15-20 seconds to mix the liquid within the wells. It is important to ensure that the blue color changes to yellow completely.

• Read the absorbance of each well at 450 nm ( using 620 to 630 nm as the reference wavelength to minimize well imperfections) in a micro plate reader. The results should be read within 30 minutes of adding the stop solution.