Anti-thyroglobulin Autoantibodies Research Articles

An enzyme immunoassay for the measurement of anti-thyroglobulin autoantibody in human serum

An enzyme-linked sandwich immunoassay using human thyroglobulin conjugated with β- d-galactosidase and silicone rods coated with human thyroglobulin was developed for the measurement of circulating anti-thyroglobulin autoantibody. The volume of serum needed for the assay was as little as 5 μ1. The sensitivity of the assay was approximately 7 × 10 −15 mol/tube of antithyroglobulin immunoglobulin G corresponding to 220 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The specificity of the assay was demonstrated by (1) parallelism of the standard curve with dilution of sample sera of patients with thyroid diseases, and (2) non-detectability of anti-thyroglobulin in autoantibody in the sera of normal subjects. The precision of the assay was proven by (1) sufficient recovery of antithyroglobulin immunoglobulin G added to serum, and (2) coefficients of variance within and between assays were 6.5 to 10.3% and 4.9 to 14.1%, respectively. No effect of thyroglobulin on the present assay was observed when the ratio of the amount of thyroglobulin to that of anti-thyroglobulin immunoglobulin G was lower than 1:10. Furthermore, a significant correlation was observed between anti-thyroglobulin autoantibody concentrations measured by our enzyme immunoassay and those by tanned red cell hemagglutination ( r = 0.78), and between those by the enzyme immunoassay and those by radioimmunoassay ( r = 0.80).

Study on Enzyme Immunoassays for the Measurement of Thyroglobulin and Anti-Thyroglobulin Autoantibody in Human Serum

Enzyme-linked sandwich immunoassays for the measurement of thyroglobulin and anti-thyroglobulin autoantibody in human serum using silicone rod and beta-D-galactosidase were studied. These methods showed excellent results in specificity, sensitivity, precision and clinical application. 1) A method using silicone rod coated with rabbit (anti-human thyroglobulin) immunoglobulin G and rabbit (anti-human thyroglobulin) monovalent fragment of immunoglobulin G (Fab') conjugated with beta-D-galactosidase was developed for the measurement of circulating thyroglobulin. The sensitivity of the assay with as little as 2 microliter of serum was 10.7 amoles/tube corresponding to 3.5 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The correlation coefficient between values determined by the present assay and a double-antibody radioimmunoassay was 0.99 (n = 63, p less than 0.001). Circulating thyroglobulin was detectable in 90% of 146 normal subjects, the concentration being 13.3 +/- 10.3 ng/ml (mean +/- S.D.). Interference of anti-thyroglobulin autoantibody with the assay for thyroglobulin was smaller than that in radioimmunoassay. 2) Another method using human thyroglobulin conjugated with beta-D-galactosidase and silicone rod coated with human thyroglobulin was developed for the measurement of circulating (anti-human thyroglobulin) autoantibody. The sensitivity of the assay with as little as 5 microliter of serum was 7 fmoles/tube corresponding to 220 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The highly significant correlation was observed between the concentrations of anti-thyroglobulin autoantibody determined by the present assay and a radioimmunoassay (r = 0.80, n = 74, p less than 0.001) and also between those by the present assay and those by tanned red cell hemagglutination (r = 0.78, n = 199, p less than 0.001). No effect of thyroglobulin on the present assay was observed unless the ratio of the amount of thyroglobulin to that of (anti-human thyroglobulin) immunoglobulin G was higher than a tenth.

Sensitive radioimmunological screening test for antithyroglobulin autoantibodies.

The test described here was designed as a screening test to be used in conjunction with the radioimmunoassay for human thyroglobulin. Anti-thyroglobulin autoantibodies, when present even in low concentrations, interfere with thyroglobulin determination. A 30-min incubation allows binding of 125I-thyroglobulin to endogenous anti-thyroglobulin autoantibodies. A subsequent 2-h incubation with goat anti-human gamma-globulin causes precipitation of such complexes. The immunoprecipitable radioactivity reflects the binding capacity of the sample for 125I-labeled thyroglobulin. Samples that are devoid of autoantibodies, and are therefore suitable for valid thyroglobulin determinations, bind less than 6% of the radiolabeled thyroglobulin. The sensitivity of this radioimmunoassay exceeds that of the tanned-erythrocyte hemagglutination test.

Radioimmunoassay for measurement of thyroglobulin in human serum.

A specific and reproducible double antibody radioimmunoassay for the measurement of thyroglobulin (HTg) in human serum has been developed. Since antithyroglobulin autoantibodies combine with the [(131)I] HTg tracer, antibody-positive sera were rejected for measurement. Specificity is demonstrated in that thyroid analogous such as thyroxine (T(4)), triiodothyronine (T(2)) monoiodotyrosine (MIT) and diiodotyrosine (DIT) did not crossreact. Sera previously reacted with anti-HTg-Sepharose contained no immunoassayable HTg. Finally, sera obtained from patients after total thyroid ablation for thyroid carcinoma did not contain demonstrable HTg. The sensitivity of the assay is 1.6 ng/ml, and HTg was detectable in 74% of 95 normal subjects. The mean concentration was 5.1 ng/ml +/-0.49 SEM (range <1.6-20.7 ng/ml). Day to day variation in HTg levels is large in some euthyroid subjects and nearly absent in others. HTg was detectable in 90% of the sera obtained in 23 pregnant women at delivery in whom a mean concentration of 10.1 ng/ml +/-1.3 SEM was observed. The mean level for the corresponding newborn infants at birth was 29.3 ng/ml +/-4.7 SEM a value significantly higher than the mean maternal HTg concentration (P <0.01). A group of 17 thyrotoxic individuals all had elevated HTg levels; the mean for this group was 344.8 ng/ml +/-90.7 SEM. In the acute phase of subacute thyroiditis HTg was also elevated in all of 12 patients, and the mean for this group was 136.8 ng/ml +/-74.6 SEM.

Radioimmunoassay for human antithyroglobulin antibodies. I. The relationship between tanned cell haemagglutination and a double antibody technique.

The present study describes a double antibody technique to evaluate antithyroglobulin autoantibodies in human serum. Rabbit anti-human &lt;i&gt;&amp;#947;G &lt;/i&gt;is used to precipitate the immunocomplexes between labelled thyroglobulin and autoantibodies. Thyroglobulin has been labelled either &lt;i&gt;in vivo, &lt;/i&gt;or chemically by electrolysis of iodide, a procedure which also produced substantial dissociation of the protein. The double antibody technique was compared with haemagglutination of sheep red blood cells coated with human thyroglobulin. A good correlation was established between the tanned red cell haemagglutination titre and the double antibody technique when ‘ &lt;i&gt;in vivo&lt;/i&gt;’ labelled thyroglobulin was employed. The use of chemically iodinated thyroglobulin, purified after labelling, increased the sensitivity of the test 100 fold so that 38% of sera from patients with thyroid diseases having negative haemagglutination titres precipitate between 15% and 65% of the labelled antigen. The sensitivity and simplicity of this method provide a useful tool in clinical as well as in experimental work.

Endemic Cretinism in Goiaz, Brazil

Twenty-six endemic cretins were studied in the middle west of Brazil, an endemic goiter region. Goiter was found in 18 patients and deaf-mutism in 10. Clinical and laboratory evidence of hypothyroidism was present in 2 cases. In all others the determination of the 24-hr I131 thyroid uptake and PBI127 gave normal results. A positive serum (low titer) for antithyroglobulin auto-antibodies (TRC test) was found in only one cretin. The clinical appearance of the patients conformed to the descriptions in classic papers on endemic cretinism. All the cretins showed characteristic neurologic signs: oligophrenia, generalized hyperreflexia, spasticity and loss of muscular strength in the lower limbs. In 10 patients we performed electroencephalograms and observed diffuse anomalies; no abnormalities were observed on pneumoencephalograms in 6 cretins. The pathogenesis of endemic cretinism is discussed.