新型人绒毛膜促性腺激素及其代谢物的世界卫生组织参考试剂的制备与特性鉴定
Preparation and characterization of new WHO reference reagents for human chorionic gonadotropin and metabolites.
作者信息
Birken Steven, Berger Peter, Bidart Jean-Michel, Weber Matthias, Bristow Adrian, Norman Rob, Sturgeon Catharine, Stenman Ulf-Håkan
机构信息
College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA.
出版信息
Clin Chem. 2003 Jan;49(1):144-54. doi: 10.1373/49.1.144.
BACKGROUND
The currently used standards for human chorionic gonadotropin (hCG) and its alpha and beta subunits (hCGalpha and hCGbeta) 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 hCGbeta (hCGbetan), which contain cuts in the peptide backbone between amino acids 44-45 or 47-48 in hCGbeta, and a fragment of hCGbeta (hCGbetacf) 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, hCGbetan, and hCGbetacf 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, hCGbeta 99/650, hCGalpha 99/720, hCGn 99/642, hCGbetan 99/692, and hCGbetacf 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.
背景
目前使用的人绒毛膜促性腺激素（hCG）及其α和β亚基（hCGα和hCGβ）标准品含有大量hCG的污染变体和其他杂质。此外，hCG及其亚基的一些部分降解形式已具有潜在的临床重要性，例如，“切口”形式的hCG（hCGn）和hCGβ（hCGbetan），其在hCGβ的氨基酸44 - 45或47 - 48之间的肽主链上有切口，以及由氨基酸6 - 40和55 - 92通过二硫键连接在一起的hCGβ片段（hCGbetacf）。国际临床化学和检验医学联合会（IFCC）任命了一个工作组，旨在制备hCG及相关物质的新标准，以改善其免疫测定的标准化。
方法
通过先前开发的纯化方法结合疏水相互作用色谱法和反相高效液相色谱法制备了大量的hCG及其亚基以及hCGn、hCGbetan和hCGbetacf。每种制剂都基于氨基酸和序列分析、碳水化合物组成和电泳图谱进行了表征。还对相关污染蛋白进行了免疫测定。
结果
主要制剂均一且不含污染蛋白。最终制剂通过氨基酸分析确定浓度。
结论
基于氨基酸分析以物质浓度（mol/L）进行校准，这些制剂将有助于改善hCG及其代谢物免疫测定的标准化。这六种制剂现已被世界卫生组织（WHO）确立为免疫测定的新的第一参考试剂，其编号如下：hCG 99/688、hCGβ 99/650、hCGα 99/720、hCGn 99/642、hCGbetan 99/692和hCGbetacf 99/708。与第三国际标准品（75/537）相比，临床上最重要的hCG参考试剂（99/688）不含hCGn且游离亚基含量可忽略不计。
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