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|Title:||Preparation and characterization of new WHO reference reagents for human chorionic gonadotropin and metabolites|
|Citation:||Clinical Chemistry, 2003; 49(1):144-154|
|Publisher:||Amer Assoc Clinical Chemistry|
|Steven Birken, Peter Berger, Jean-Michel Bidart, Matthias Weber, Adrian Bristow, Rob Norman, Catharine Sturgeon and Ulf-Håkan Stenman|
|Abstract:||Background: The currently used standards for human chorionic gonadotropin (hCG) and its and ß subunits (hCG and hCGß) contain substantial amounts of contaminating variants of hCG and other impurities. Furthermore, some partially degraded forms of hCG and its subunits have become of potential clinical importance, e.g., "nicked" forms of hCG (hCGn) and hCGß (hCGßn), which contain cuts in the peptide backbone between amino acids 44–45 or 47–48 in hCGß, and a fragment of hCGß (hCGßcf) consisting of amino acids 6–40 and 55–92 bound together by disulfide bridges. The IFCC appointed a working group with the aim of preparing new standards for hCG and related substances to improve standardization of their immunoassays. Methods: Large amounts of hCG and its subunits as well as of hCGn, hCGßn, and hCGßcf were prepared by previously developed purification methods in combination with hydrophobic interaction chromatography and reversed-phase HPLC. Each preparation was characterized on the basis of amino acid and sequence analyses, carbohydrate composition, and electrophoretic patterns. Immunoassays for relevant contaminating proteins were also performed. Results: The major preparations were homogeneous and free of contaminating proteins. Concentrations of the final preparations were determined by amino acid analysis. Conclusions: Calibrated in substance concentrations (mol/L) based on amino acid analyses, these preparations will facilitate improved standardization of immunoassays for hCG and its metabolites. The six preparations have now been established by the WHO as new 1st Reference Reagents for immunoassays with the following codes: hCG 99/688, hCGß 99/650, hCG 99/720, hCGn 99/642, hCGßn 99/692, and hCGßcf 99/708. In contrast to the 3rd International Standard (75/537), the clinically most important Reference Reagent for hCG (99/688) contains no hCGn and negligible amounts of free subunits.|
|Keywords:||Humans; Chorionic Gonadotropin; Protein Subunits; Immunoassay; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Isoelectric Focusing; Reference Standards; World Health Organization|
|Rights:||© 2003 American Association for Clinical Chemistry, Inc.|
|Appears in Collections:||Obstetrics and Gynaecology publications|
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