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Chapter 120 : Endocrinopathies
This chapter describes the various procedures currently used to detect circulating autoantibodies in patients with endocrine disease, i.e., thryoiditis, Graves’ disease, insulin-dependent diabetes mellitus, Addison’s disease, and pernicious anemia. The general method of indirect immunofluorescence (IF) is the screening test used most frequently to detect tissue- or organ-specific autoantibodies. Commercial hemagglutination kits for thyroglobulin antibodies are less sensitive than the tanned-cell hemagglutination (TCH) test described. Indirect IF performed on sections of human or monkey thyroid can demonstrate antibodies to thyroglobulin, CA2, and microsomes of thyroid epithelial cells, and antinuclear antibodies. The most commonly used tests for determining thyroid peroxidase (TPO) antibodies are indirect IF and commercially available hemagglutination assays or ELISAs. Separate diabetes mellitus categories consist of gestational diabetes and ‘’specific types of diabetes’’ usually with defined mutations or accompanying pathology (e.g., pancreatic abnormalities, endocrinopathies, or drug induced). The current insulin autoantibodies (IAA) assay is not able to distinguish between natural IAA and induced insulin antibodies. The current major format for determination of islet autoantibodies uses fluid-phase radioimmunobinding assays, applied to all three major islet autoantibody tests, including glutamic acid decarboxylase (GAD) autoantibodies (GAA), ICA512AA, and IAA. The majority of sera from type 1 diabetes patients target more than two epitopes on ICA512 molecules, while non-disease-related antibodies usually react with only one epitope. Anti-islet autoantibodies find their primary application in the differential diagnosis of the forms of diabetes and in predicting risk of progression to overt diabetes.
Key Concept Ranking
- Enzyme-Linked Immunosorbent Assay
Overall correlation between the CCH test and ELISA. Each dot represents the mean ELISA value for the group of sera with the individual CCH titers.
Indirect IF for antibodies to thyroid microsomes with an unfixed, air-dried section of monkey thyroid. Only the cytoplasm of the thyroid epithelial cells is stained. (Magnification, ×80.)
Indirect IF for antibodies to adrenal cortex, with an unfixed, air-dried section of monkey adrenal cortex. All layers of the adrenal cortex are stained. (Magnification, × 32.)
Same as Fig. 3 but at a higher magnification. Only the cytoplasm of the adrenal cells is stained. (Magnification, ×80.)
Indirect IF for antibodies to parietal cells of the gastric mucosa, with an unfixed, air-dried section of rat stomach. The cytoplasm of most cells is stained. (Magnification, ×100.)
TSAb-induced cAMP accumulation in FRTL5 and CHO-TSH-R cells incubated in isotonic medium. Cells were incubated for 2 h in the absence (control) or presence of the indicated concentrations of TSAb (purified IgG), and cAMP was measured in cell extracts.
TBI assay with a commercial kit (Kronus, Inc., Boise, Idaho). Dilutions were carried out with pooled serum from healthy humans. A, B, C, and D refer to sera from mothers, all on replacement therapy, who had hypothyroid neonates. TSAb was from a mother who was treated with 131I for hyper-thyroidism of Graves’ disease and subsequently had two hyper-thyroid neonates. (Reprinted, with permission of The Endocrine Society, from M. Zakarija, J. M. McKenzie, and M. S. Eidson, Transient neonatal hypothyroidism: characterization of maternal antibodies to the thyrotropin receptor, J. Clin. Endocrinol. Metab. 70:1239-1246, 1990.)
Assays with CHO-TSH-R cells in a low-salt medium. (A) Inhibitory effect of TBAb on TSAb- and TSH-induced accumulation of cAMP. (B) Biphasic effect of a patient’s IgG (IgG-Z). Cells were incubated for 2 h with the test substances (TRAb as purified IgG) and concentrations indicated, and cAMP in the medium was measured.
Progression to overt type 1A diabetes of first-degree relatives of patients with type 1A diabetes (DM) relative to number of anti-islet autoantibodies expressed (autoantibod-ies to GAD65, ICA512, and insulin). ♦, three antibodies; ▪, two antibodies; ▴, one antibody. (From http://www.barabaradavis-center.org, Immunology of Diabetes.)
The current well-identified anti-islet autoantibody assays. (From http://www.barbaradaviscenter.org, Immunology of Diabetes.) Islet autoimmunity is defined as one or more autoanti-bodies persistent for at least 3 to 6 months.
General outline of fluid-phase 96-well plate assays for autoantibodies. In this example 125I-insulin is utilized, but the format is identical for GAD65 and ICA512 assays.
Standard curve for calculation of WHO units. The x axis shows the counts per minute from the assay after removing the background counts per minute, and the y axis shows known log2 WHO units.
Biochemically characterized autoantigens