Anti-Thyroid EIA

Kallestad™ Anti-Thyroglobulin (Anti-Tg) Autoimmune EIA

Clinical Utility

Thyroglobulin (Tg), thyroid peroxidase (TPO), and the TSH receptor are considered the major autoantigens associated with chronic autoimmune thyroiditis. Clinical diagnosis is usually based on the presence of serum or plasma autoantibodies to Tg and TPO. In Graves' disease (chronic primary hyperthyroidism), the TSH receptor is the antigen most directly involved in clinical manifestations of the disease.¹

Anti-Tg autoantibodies are predominantly of the IgG class. They are detected, often with TPO autoantibodies, in the majority of cases of Graves' disease and Hashimoto's thyroiditis, including other variants of chronic primary hypothyroidism such as myxoedema and asymptomatic thyroiditis. They have also been detected in spontaneous or postpartum painless thyroiditis, in thyroid autoimmunity with rheumatoid arthritis, and in non-thyroid autoimmune diseases such as Addison's disease and type 1 diabetes mellitus.

Reference

1. Rapoport B. Exp Clin Endocrinol. 97 (2/3):147–152, 1991.

Kallestad™ Anti-Thyroglobulin (Anti-Tg) Autoimmune EIA

Pre-Test Setup Instructions

Step 1. Bring all reagents to room temperature (18–25°C) 30 minutes before use. Step 2. Prepare the wash solution.
Step 3. Prepare sample diluent solution.
Step 4. Make 1:100 working solutions by diluting 10 µL of patient's serum and positive/negative controls.

Test Procedure

See Procedure Notes and Pre-Test Setup Instructions.

• Qualitative Protocol — Run Reference Control, Positive/Negative Controls, and Samples
• Quantitative Protocols — Run Standards (0–4) Positive/Negative Controls, and Samples

Step 1. Apply 100 µL diluted patient samples, controls and standards to wells. Step 2. Incubate wells for 60 ± 5 minutes at room temperature (18–25°C).
Step 3. Wash wells 5x with ~200 µL of wash solution.Remove all liquid before proceeding.
Step 4. Apply 100 µL HRP conjugate to all wells.
Step 5. Incubate wells for 30 ± 5 minutes at room temperature (18–25°C). Step 6. Wash wells 5x with ~200 µL of wash solution. Remove all liquid before proceeding. Step 7. Develop color by adding 100 µL of substrate to each well.
Step 8. Incubate for 30 ± 5 minutes at room temperature (18–25°C). Step 9. Stop color development by adding 100 µL of stop solution to each well. Step 10. Read results within one hour with an EIA reader set to 550 nm.

Kallestad™ Anti-Thyroglobulin (Anti-Tg) Autoimmune EIA

Result Interpretation

Kallestad™ Anti-Thyroid Peroxidase (Anti-rTPO) Autoimmune EIA

Clinical Utility

Autoimmune thyroid disorders encompass autoimmune destruction and stimulation; both states are associated with predominantly IgG local and circulating thyroid autoantibodies.

Trotter et al.¹ and Roitt et al.² showed that many patients with advanced thyroiditis had antibodies for a thyroid antigen distinct from Tg (thyroglobulin). This was termed thyroid microsomal antigen (TMA). Considerable evidence indicates that TMA is antigenically related to thyroid peroxidase (TPO), a membrane-bound glycoprotein enzyme with an approximate mass of 101 kD, whose in vivo function is the iodination of tyrosine in the synthesis of the thyroid hormones T3 and T4. Anti-TPO antibodies are found, often in conjunction with anti-thyroglobulin autoantibodies, in the majority of cases of Hashimoto's thyroiditis and Graves' disease and in cases of primary myxoedema. The relationship of autoimmune thyroid disease in pregnancy has been the subject of considerable interest, with the demonstration of TPO antibodies in most cases of post-partum thyroid disease syndrome^3–6 and the association of thyroid autoantibodies with increased miscarriage risk.⁷ Anti-TPO antibodies are found in other non-thyroid conditions, e.g. pernicious anemia,^8,9 diabetes mellitus,^10,11 rheumatoid arthritis,¹² Addison's disease¹¹ and Sjögren's syndrome.⁹ In addition, anti-TPO antibodies are detectable at low levels in 2–8% of apparently healthy individuals, particularly in the elderly and more often in women than in men, although the clinical significance of this is unclear.

Bio-Rad's new recombinant kit format maintains the same Bio-Rad Kallestad EIA methodology, calibration to NIBSC 66/387 thyroid microsomal antibody reference preparation, and kit assay protocol (60'/30'/30' incubation) as the rest of the Bio-Rad Kallestad autoimmune EIA product range.

This assay provides reproducible results through the use of DNA recombinant technology. This technology minimizes the potential of antisera to bind both to specific and unspecific thyroid cell components. The Bio-Rad Kallestad Anti-Thyroid Peroxidase Kit is made with human recombinant TPO antigen, which has been demonstrated to be highly specific for TPO autoantibodies. Additional features include a lower clinical cut-off, reflecting increased sensitivity and specificity, and a narrower measuring range, ensuring greater accuracy and precision.

References

1. Trotter WR, et al. Proc R Soc Med. 50:961, 1957.
2. Roitt IM, et al. Lancet. 2:1027–1033, 1958.
3. Vargas VT, et al. J Clin Endocrin Metab. 67:327–333, 1988.
4. Jansson R, et al. J Clin Endocrin Metab. 58:681–687, 1984.
5. Stagnaro-Green A. Thyroid Today. 16:1–11, 1993.
6. Lazarus JH. Thyroid. 9 (7):685–689, 1999.
7. Stagnaro-Green A, et al. Jama. 264 (11):1422–1425, 1990.
8. Estienne V, et al. Eur J Endocrinol. 141:563–569, 1999.
9. Feldt-Rasmussen, et al. Autoimmunity. 9:245–253, 1991.
10. Chang CC, et al. European Journal of Endocrinol. 139:44–48, 1998.
11. Scherbaum WA. Acta Endocrinol (Copenh) Suppl. 281:325–329, 1987.
12. Walker DJ, et al. Ann Rheum Dis. 45:323–326, 1986.

Kallestad™ Anti-Thyroid Peroxidase (Anti-rTPO) Autoimmune EIA

Result Interpretation