International Reference Preparation of Human Chorionic Gonadotrophin for Immunoassay: Potency Estimates in Various Bioassay and Protein Binding Assay Systems; And International Reference Preparations of the Α and Β Subunits of Human Chorionic Gonadotrophin for Immunoassay

Abstract

The preparation and nature of the International Reference Preparation of Human Chorionic Gonadotrophin (HCG) for Immunoassay (IRP), as well as that of a second batch of ampoules (HCG 75/589) prepared identically from the same HCG preparation, are described.A collaborative study of these materials was carried out by 11 laboratories in eight countries, using different bioassay and immunoassay methods. Using the various in-vivo and in-vitro bioassays and receptor assays, the mean log potency estimates for each method within each laboratory of the HCG content of ampoules of the IRP, in terms of the Second International Standard of Human Chorionic Gonadotrophin for Bioassay (IS), were homogeneous and gave an overall weighted geometric mean (95% confidence limits) of 650 (632\p=n-\669) International Units (i.u.)/ampoule. There was considerable heterogeneity of potency estimates of the IRP in terms of the IS both within and between many of the immunoassay systems (reflecting the impurity of the IS), and hence attempts to calibrate the IRP with immunoassay systems of different specificities were invalid.Immunoassay estimates of the HCG content of preparations of serum and urine, in terms of the IRP, showed considerable heterogeneity between assay systems (although the degree of this heterogeneity was no greater than that observed using the IS as standard), but the ranking order between preparations was consistent.Confirmation was obtained that contamination of the IRP with HCG-\g=a\ and HCG-\g=b\ subunits was insignificant. Accelerated degradation studies of the IRP stored at increased temperatures suggested that its stability under normal storage conditions would be satisfactory.It was agreed that the IRP was suitable to serve as an international reference preparation for immunoassay, and it was assigned a unitage of 650 i.u./ampoule on the basis of bioassay calibration.Since the ampoules of HCG (75/589) did not differ significantly from the IRP in any of the assay systems studied, it appeared to be equally suitable as a reference preparation.The International Reference Preparations of the \ g=a\ and \ g=b\ Subunits of Human Chorionic Gonadotrophin for Immunoassay are also described.

Designing novel nano-immunoassays: antibody orientation versus sensitivity

“…The concentration of hCG used is expressed in international units (IU) per mL. By definition 1 IU of hCG is equal to 0.11 μg of hormone (~ 3.0 pmol) [36].…”

Preparation and Characterization of New WHO Reference Reagents for Human Chorionic Gonadotropin and Metabolites

“…the 3rd International Standard (IS) for hCG (coded 75/ 537) and the 1st IRPs for its dissociated subunits, hCG␣ (75/569) and hCG␤ (75/551) (2,3 ). The recently established 4th IS (75/589) was prepared at the same time as the 3rd IS from the same batch and is essentially identical to the 3rd IS, differing only by two separate filling procedures (3 ).…”

“…The recently established 4th IS (75/589) was prepared at the same time as the 3rd IS from the same batch and is essentially identical to the 3rd IS, differing only by two separate filling procedures (3 ).…”

“…These hCG metabolites, which are found mainly in urine but may also occur in serum (4 -7 ), are of interest both because of their variable reactivity in existing assays and their potential clinical utility, particularly in urine assays (1, 5, 8 -10 ). Substantial amounts of these metabolites, as well as traces of protease activity, have been shown to be present in the current standard preparations for hCG (IS 75/537 and 75/589), hCG␣ (IRP 75/569), and hCG␤ (IRP 75/551) (3,11,12 ), and these contaminants may be partly responsible for variability in the behavior of the 3rd/4th IS in various commercial hCG assay systems (13,14 ). Contamination with hCGn is also a major problem because some hCG assays recognize hCGn poorly, whereas others recognize hCGn and hCG equally, potentially giving rise to calibration errors.…”

The beta-subunit of human chorionic gonadotrophin exists as a homodimer

“…The international reference preparation of free hCG , CR123, which is derived from splitting intact heterodimeric hCG isolated from pregnancy urine, was a kind gift from Dr Steven Birken (Columbia University, New York, USA) (Storring et al 1980). MALDI matrices -cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), 3,5-dimethoxy-4-hydroxybenzoic acid (sinapinic acid (SA)) and 5-methoxysalicylic acid (MSA) were purchased from Aldrich Chemical Co. (Gillingham, Dorset, UK) and used without further purification.…”