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A T A B

Direct ¹²⁵I-Immunoradiometric assay kit for the quantitative determination of serum anti-thyroglobulin auto-antibodies.

Cat. BL-06-100

for in vitro diagnostic use only

Summary and background of the test:

Immunological action on the cells of the thyroid gland can either result in stimulating or suppressing thyroid activity (1).Auto-antibodies to several distinct thyroid antigens, including thyroglobulin (TG), thyroid microsomal antigen, and the TSH receptor, are often present in the sera of patients with hyperthyroid Graves' disease. This disorder may include a peculiar condition known as exophthalmos. Thyroid hormones are produced in excess (2).In cases of Hashimoto's goitre, antibodies to thyroglobulin are synthesized by immunocytes invading the thyroid gland, diminishing its function that may disappear entirely, if untreated. Similarly, patients with autoimmune hypothyroidism often have serum antibodies to thyroid microsomal antigen (3), identified as thyroid peroxidase (TPO) (4 ® 11).The most widely used method for the detection of circulating auto-antibodies has been passive haemagglutination. However, this technique allows only semi-quantitative determination, and its sensitivity may be inadequate for special investigation purposes.Different techniques are presently used to detect and quantify auto-antibodies to thyroglobulin and thyroid peroxidase : antigen-coated plates (12), antigen-coated tubes (13), monoclonal antibody-coated tubes (14), and direct binding of ¹²⁵I-labelled antigens to corresponding auto-antibodies (15).The present assay system is based on the direct interaction between ¹²⁵I-labelled Tg and auto-antibodies.Both design of the kit and standardization against MRC anti- thyroglobulin antibody research standard A (65/93) result in improved sensitivity, precision, and ease of handling.

Principle of the test :

If present, anti-thyroglobulin auto-antibodies from the samples will bind ¹²⁵I-labelled human TG. Simultaneously, IgG-h-TG complexes will be precipitated by Protein A, exhibiting a strong affinity for the Fc-chain of most mammalian immunoglobulins and their antigen complexes.

The activity of the precipitate will be directly related to the levels of the anti-TG antibodies initially present.

The level of unknown auto-antibodies is then determined by comparing the radioactivity of the isolated precipitate with data established using known standards in the same assay system.

Results are expressed in IU/ml, calibrated on the international reference preparation of human anti-Thyroglobulin serum (MRC 65/93).

Materials provided

Kit contains sufficient reagents for 100 determinations.

1. Anti-TG Ab standards & control : 7 vials containing each 500 μl. Control: 150 + 25 IU/ml.

Standards: 0-100-300-1000-3000-10000 IU/ml.

2. ¹²⁵I-TG tracer : 2 vials (blue solution) containing each 5.2 ml. Ready for use.

Activity per vial £ 3μCi or £ 111 kBq.

3. Protein A : 2 vials containing each 10.5 ml ready-to-use blue suspension (Staphylococcus aureus cells).

4. Diluent : 1 vial containing 60 ml of ready-to-use green-colored solution.

All reagents are ready for use and should be stored at 2^o - 8^o C.

Refer to the expiration date on the kit label for stability.

Specimen collection and preparation:

Sera should be separated from blood cells immediately after collection. Sera are stable for at least 7 days at 4^o C and for longer periods of time when stored frozen.

Pre-dilution of patient samples:

Before analysis, samples must be diluted 1/20 in the diluent as follows:

25 μl samples + 500 μl Diluent (green).

Do not attempt to dilute the standards and control serum; they are already diluted and ready for use.

Assay procedure:

Bring reagents to room temperature and mix before use. Label normal or conical polystyrene disposable tubes for total counts (Tc), standards, control sera and unknowns.

1. Pipet 50 μl of standards and control, 1/20 pre-diluted samples into the corresponding tubes.

2. Add 100 μl of tracer solution (orange) to each tube.

3. Mix and incubate for 1 hour at room temperature.

4. Add 200 μl of Protein A to each tube, except Tc. Maintain moderate magnetic stirring of the suspension during the transfer.

5. Mix and incubate for 10-15 minutes at room temperature.

6. Add 2 ml of NaCl 9 ‰ to each tube, except Tc. Centrifuge all tubes at 4° C, except Tc, for 10-15 minutes at 1300-1500g.

7. Decant all tubes as a whole, discarding the supernatant into a radioactive waste container. While tubes are inverted, gently blot the final drops onto absorbent paper. Do not aspirate. Do not let stand inverted.

When using conical tubes, an aspiration of the supernatant should be possible, using a suction device with the needle adjusted so that it is just 2 mm above the visible precipitate.

8. Record the counts per minute (cpm) for each tube. Count all tubes for one minute.

ATAB Flow chart.

Tubes Reagents	Tc	Standards	control	1/20diluted samples
Standards /samples	-	50 μl	50 μl	50 μl
Tracer	100 μl	100 μl	100 μl	100 μl
Mix and incubate at room temperature for 1 hour
Protein A	-	200 μl	200 μl	200 μl
Mix and incubate at room temperature for 10-15 minutes
NaCl 9 ‰	-	2 ml	2 ml	2 ml
Centrifuge at 4° C during 15 minutes and discard supernatants
Count in a gamma counter

Data table (Example).

#	Duplicate cpm		Mean cpm	%B/Tc	Conc. IU/ml
Tc Zero Std 100 Std 300 Std 1000 Std 3000 Std 10000 Control Sample 1	63 957 618 5 546 14 152 28 197 39 879 47 087 8 364 31 532	65 196 623 5 623 14 053 28 173 40 131 47 096 8 331 31 559	64 571 621 5 585 14 103 28 185 40 005 47 092 8 348 31 546	100 % 0.9 % 8.6 % 21.6 % 43.6 % 61.4 % 72.4 % 12.9 % 48.7 %	155 1340

Calculation of results:

Data need not be expressed as counts per minute (cpm) but the counting period must be the same for all tubes that are counted.

Determine the average counts for each set of duplicate tubes. Divide this value by the average net counts of Tc, and multiply by 100 to yield the % B/Tc.

% B/Tc = cpm (Stds, Control or samples) x 100/cpm (Tc)

Plot % B/Tc for each standard vs its concentration in IU/ml. The concentration of anti-TG in the unknown samples may be read directly from the standard curve.

Samples exhibiting titers greater than 10 000 IU/ml should be more diluted using the diluent. If necessary, multiply by the dilution factor to determine the initial concentration.

Expected Values:

Each laboratory should establish its own normal range.

Anti-TG values Interpretation

£ 100 IU/ml Negative

100-200 IU/ml Gray zone

³ 200 IU/ml Positive

Bibliography:

1. Pinhera A, Fenzi GF, Bartalena L, Chiovato L, Marcocci C, Baschieri L. Antigen-antibody systems involved in thyroid autoimmunity. In: Pinchera A, Doniach, Fenzi G, Baschieri L, eds. Autoimmune aspects of endocrine disorders. London: Academic Press, 1980; 57-72.

2. Doniach, D., Humoral and genetic aspects of thyroid autoimmunity. Clin. Endocrinol. Metab. 4, 267-285 (1975).

3. Doniach D, Bottazzo G F & Russel R C G (1979): Goitrous autoimmune thyroiditis. Clin Endocrinol Metab 8:63-80.

4. Czarnocka B, Ruf J, Ferrand M, Carayon P. Antigenic relationship between thyroid peroxidase and the microsomal antigen involved in thyroid autoimmune thyroid diseases. C R Acad Sci (Paris) 1985; 300:577-80.

5. Czarnocka B, Ruf J, Ferrand M, Carayon P,Lissitzky S. Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases. FEBS lett 1985:147-52.

6. Czarnocka B, Ruf J, Ferrand M,Lissitzky S, Carayon P. Interaction of the highly purified thyroid peroxidase with anti-microsomal antibodies in autoimmune thyroid diseases. J Endocrinol Invest 1986; 9:135-8.

7. Portmann L, Hamada N, Heinrich G, DE Groot LJ. Anti-thyroid peroxidase antibody in patients with autoimmune thyroid disease: possible identity with anti-microsomal antibody. J Clin Endocrinol Metab 1985; 61:1001-3.

8. Kotani T, Umeki K, Matsunaga S, Kato E, Ohtaki S. Detection of autoantibodies to thyroid peroxidase in autoimmune thyroid diseases by micro-ELISA and immunoblotting. J Clin Endorinol Metab 1986;62:928-33.

9. Nakajima Y, Howells RD, Pegg C, Jones ED, Smith BR. Structure-activity analysis of microsomal antigen/thyroid peroxidase. Mol Cell Endocrinol 1987; 53:15-23.

10. Ruf J, Czarnocka B, De Micco C, Dutoit C, Ferrand M, Carayon P. Thyroid peroxidase is the "microsomal" autoantigen involved in thyroid autoimmunity. Acta Endocrinol (Copenhagen) 1987;Suppl 281:49-56.

11. Mariotti S, Anelli S, Ruf J, et al. Comparison of serum thyroid microsomal and thyroid peroxidase autoantibodies in thyroid diseases. J Clin Endocrinol Metab 1987;65:987-93.

12. Schardt CW, McLachlan SM, Matheson J, Rees Smith B. An enzyme-linked immunoassay for thyroid microsomal antibodies. J Immunol Methods 1982;55:155-68.

13. Furmaniak J, Bradbury J, Rees Smith B. Antibodies to membrane antigens in autoimmune thyroid disease. Acta Endocrinol 1987;116:13-20.

14. Ruf J, Czarncka B, Ferrand M, Doullais F, Carayon P. Novel routine assay of thyroperoxidase autoantibodies. XClin Chem 1988;34:2231-4.

Very important notice for export

Since 1rst of January 2012

The production and the export of the clinical laboratory diagnostic kits RIA and ELISA

are transferred to the company : Asbach Medical Products GmbH

Tel: + 49 62 62 91 74 02 - Fax: + 49 62 62 91 76 99 - Email: info@amp-asbach.com

If any problem please do not hesitate to contact us.

Last updating 14/12/11