Preparation and characterization of new WHO reference reagents for human chorionic gonadotropin and metabolites.
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.
Birken
Steven
S
College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA. sb18@columbia.edu
Berger
Peter
P
Bidart
Jean-Michel
JM
Weber
Matthias
M
Bristow
Adrian
A
Norman
Rob
R
Sturgeon
Catharine
C
Stenman
Ulf-Håkan
UH
D015153:Blotting, Western; D006063:Chorionic Gonadotropin / Q000302:isolation & purification / Q000378:metabolism* / Q000592:standards*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007118:Immunoassay; D007525:Isoelectric Focusing; D021122:Protein Subunits; D012015:Reference Standards; D014944:World Health Organization
D006063:Chorionic Gonadotropin; D021122:Protein Subunits