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A radioligand-binding assay for detecting antibodies specific for proinsulin and insulin using 35S-proinsulin. 2003

Silvina N Valdez, and Rubén F Iacono, and Anabel Villalba, and Alejandro Cardoso Landaburu, and Mario R Ermácora, and Edgardo Poskus
School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina.

A new radioligand-binding assay (RBA) is described for the detection of insulin/proinsulin-specific antibodies using 35S-labeled proinsulin produced by a cell-free reticulocyte extract. Direct use of the crude expression product in the RBA was not feasible because the protein failed to fold properly (or had incorrectly paired disulphide bridges) and purification was hindered by interfering by-products. A refolding protocol and a chromatographic procedure were devised that readily allowed production of purified and immunochemically competent 35S-labeled proinsulin. The new RBA was compared with the reference test, in which the tracer was standard 125I-insulin. The analysis included sera from 41 diabetic patients and 25 healthy controls. Twenty-six (63.4%) and 29 (70.7%) patients scored positive by RBA using 35S-PI and 125I-insulin, respectively. The methods showed a satisfactory correlation with r(2)=0.77 and a slope not significantly different from unity (m=1.16+/-0.10; 95% confidence interval). Since the nuclide used in the assay is 35S, the procedure is compatible with standard assays for GADA and IA-2A, and thus may permit combined assays for the major early markers of autoimmune diabetes.

UI MeSH Term Description Entries
D007328 Insulin A 51-amino acid pancreatic hormone that plays a major role in the regulation of glucose metabolism, directly by suppressing endogenous glucose production (GLYCOGENOLYSIS; GLUCONEOGENESIS) and indirectly by suppressing GLUCAGON secretion and LIPOLYSIS. Native insulin is a globular protein comprised of a zinc-coordinated hexamer. Each insulin monomer containing two chains, A (21 residues) and B (30 residues), linked by two disulfide bonds. Insulin is used as a drug to control insulin-dependent diabetes mellitus (DIABETES MELLITUS, TYPE 1). Iletin,Insulin A Chain,Insulin B Chain,Insulin, Regular,Novolin,Sodium Insulin,Soluble Insulin,Chain, Insulin B,Insulin, Sodium,Insulin, Soluble,Regular Insulin
D007457 Iodine Radioisotopes Unstable isotopes of iodine that decay or disintegrate emitting radiation. I atoms with atomic weights 117-139, except I 127, are radioactive iodine isotopes. Radioisotopes, Iodine
D011384 Proinsulin A pancreatic polypeptide of about 110 amino acids, depending on the species, that is the precursor of insulin. Proinsulin, produced by the PANCREATIC BETA CELLS, is comprised sequentially of the N-terminal B-chain, the proteolytically removable connecting C-peptide, and the C-terminal A-chain. It also contains three disulfide bonds, two between A-chain and B-chain. After cleavage at two locations, insulin and C-peptide are the secreted products. Intact proinsulin with low bioactivity also is secreted in small amounts.
D011498 Protein Precursors Precursors, Protein
D011869 Radioligand Assay Quantitative determination of receptor (binding) proteins in body fluids or tissue using radioactively labeled binding reagents (e.g., antibodies, intracellular receptors, plasma binders). Protein-Binding Radioassay,Radioreceptor Assay,Assay, Radioligand,Assay, Radioreceptor,Assays, Radioligand,Assays, Radioreceptor,Protein Binding Radioassay,Protein-Binding Radioassays,Radioassay, Protein-Binding,Radioassays, Protein-Binding,Radioligand Assays,Radioreceptor Assays
D003920 Diabetes Mellitus A heterogeneous group of disorders characterized by HYPERGLYCEMIA and GLUCOSE INTOLERANCE.
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000906 Antibodies Immunoglobulin molecules having a specific amino acid sequence by virtue of which they interact only with the ANTIGEN (or a very similar shape) that induced their synthesis in cells of the lymphoid series (especially PLASMA CELLS).
D013462 Sulfur Radioisotopes Unstable isotopes of sulfur that decay or disintegrate spontaneously emitting radiation. S 29-31, 35, 37, and 38 are radioactive sulfur isotopes. Radioisotopes, Sulfur

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