Biomedical subjects
Publications and source records attributed to H Domek.
Human thyroglobulin pretreated with iodine (1mM) at alkaline pH is split to small molecular weight fragments after reduction with dithiothreitol. Iodine pretreatment alone did not induce any changes in the thyroglobulin molecular weight.
The influence of iodine-iodide solution on the biochemical and immunological properties of human thyroglobulin (hTg) were studied. Human Tg preincubated with the iodine-iodide solution is split to small molecular mass fragments after disulphide bridge reduction with dithiothreitol. The peptide bond cleavage by iodine pretreatment and reduction is possibly linked with the coupling reaction of diiodotyrosyl residues. Pretreatment of hTg with iodine-iodide solution at 1-10 microM decreased the binding of autoantibodies to hTg. The iodine-iodide induced inactivation of hTg autoepitopes is pH dependent and is possibly caused by iodination of tyrosyl residues present in the epitope structure.
Solid phase immunoenzymatic methods (ELISA) have been developed for the determination of antithyroglbulin (ATG), antimicrosomal (AMc) and antimembrane (ATMA) antibodies in blood serum. The results have been expressed in international units (IU). The level of nonspecific reaction was determined on the basis of 30 samples of blood serum obtained from healthy donors. The double standard deviation values amounted to 8 IU for antithyroglobulin antibodies, 17 IU for antimicrosomal antibodies and 53 IU for antimembrane antibodies at the serum dilution of 1:100. The values of double standard deviation obtained for the healthy donors correspond to the borderline between the positive serum samples and those containing no autoantibodies. The level of autoantibodies in patients with autoimmune diseases of the thyroid varied considerably ranging from complete absence to several thousand units per milliliter in single cases. Antithyroglobulin antibodies were determined simultaneously by using the described method and the commercial kit (Walker, Cambridge) and the results obtained by the two methods were compared. A linear correlation with the correlation coefficient r = 0.93, p < 0.001 was obtained. A good but nonlinear correlation was demonstrated with the methods expressing the results in titre values.
The effect of disulphide bridges reduction of the microsomal antigen (Mic-Ag) and thyroid peroxidase (TPO) by dithiotreitol (DTT) has been investigated. The reaction of all 67 tested sera from untreated hyperthyroid Graves' and from 22 Hashimoto's patients with high microsomal antibodies (aAb) titer was diminished by 90-95% by DTT, at pH 9.6. The remaining 5-10% of the activity was not destroyed by DTT. The residual Mic-Ag after DTT reduction was able to inhibit the binding of all 45 Graves' and 22 Hashimoto's tested aAb's to the native microsomal antigen by 100% at high concentration. Reaction of affinity purified TPO with two monoclonal antibodies (mAb) were diminished by 80% to 95% by DTT pretreatment, while the reaction of one mAb with TPO was only slightly affected. The reaction of TPO and Mic-Ag with rabbit polyclonal anti-TPO serum (rabbit a TPO) was diminished by 60% by DTT pretreatment. The immunological reactivity of TPO with aAb's was diminished by 65% after DTT pretreatment. The microsomal antigen-aAb's complex was not destroyed by DTT. Results presented in this paper suggest conformational epitope structure of the Mic-Ag recognized by aAb's in patients with thyroid autoimmune disease (AITD).
High sensitivity of the microsomal antigen epitopes recognized by autoantibodies in sera of patients with thyroid autoimmune diseases to disulphide bridge reducing agents have been detected. The shape of the inactivation curve is different for sera obtained from different patients, but for most sera 50% inactivation is obtained below 1 mM of dithioerythritol. The inactivation strongly depends on the pH of the incubation buffer and is most pronounced at alkaline pH (9.6). Cross-linking of the soluble microsomal antigen by glutaraldehyde makes the epitopes resistant to DTE. Binding of the microsomal antigen to polystyrene makes it up to 5 times more resistant to DTE. The microsomal peroxidase enzymatic activity is destroyed by low concentration of dithioerythritol and the inactivation is not dependent on the buffer pH (7.4-9.6). The data demonstrated that the major microsomal antigen epitope is discontinuous (conformation dependent).
Autoantibodies to different thyroid cell components in serum of patients with thyroid disease have been estimated by several methods. The TSH receptor antibodies (TRAb) have been detected in 77% of untreated Graves-Basedow, in 70% of thionamide drugs treated Graves-Basedow patients up to four months of the treatment and in 48% of patients with Hashimoto's thyroiditis. Microsomal antibodies (MAb) and thyroid plasma membrane antibodies (ATMA) have been detected in 84% and 68% respectively, in thionamide drugs treated Graves-Basedow and in 91% and 72% in Hashimoto's thyroiditis. The thyroglobulin antibodies have been detected in 42% of untreated Graves-Basedow, in 47% of thionamide drugs treated Graves-Basedow and in 77% of Hashimoto's thyroiditis. In 19% to 21% of patients with toxic nodular goiter underlying autoimmunological process have been established by the detection of high levels of serum autoantibodies (ATMA and MAb). In the non-toxic nodular goiter, non-thyroid autoimmune diseases and healthy blood donors only thyroglobulin antibodies were detected in a significant number of patients. Other types of antibodies (ATMA or MAb) were detected only in sporadic cases of non-thyroid autoimmune diseases.
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