| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 College of Physicians and Surgeons of Columbia University, New York, NY 10032.
2
Institute for Biomedical Aging Research, Austrian Academy of Sciences, A6020 Innsbruck, Austria.
3
Department of Clinical Biology, Institut Gustave-Roussy, 94805 Villejuif Cedex, France.
4
Klinikum Grosshadern, University of Munich, D-81377 Munich, Germany.
5
National Institute of Biological Standards and Control, Potters Bar, Herts EN6 3QG, United Kingdom.
6
Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia.
7
Department of Clinical Biochemistry, Royal Infirmary, Edinburgh EH3 9YW, United Kingdom.
8
Department of Clinical Chemistry, Helsinki University Central Hospital, Helsinki 00290, Finland.
aAuthor for correspondence. Fax 212-305-1599; e-mail sb18{at}columbia.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(75/569) and hCGß (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).
In addition to preparing improved replacements for the existing standards, the WG undertook preparation of three new standards for urinary metabolites of hCG, namely the ß-core fragment of hCGß (hCGßcf) and nicked forms of hCG (hCGn) and hCGß (hCGßn). These hCG metabolites, which are found mainly in urine but may also occur in serum (4)(5)(6)(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)(9)(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 hCGß (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. Recent advances in purification technologies provided an opportunity to prepare standards essentially devoid of these contaminants (10)(15)(16). The structural characteristics of the various forms of hCG that were to be prepared, together with their approved IFCC nomenclature, are shown in Fig. 1
.
|
This report describes the preparation and chemical characterization of the new Reference Reagents (RRs) for immunoassay standardization of hCG and hCG-related variants. Detailed data on the purification procedure and protein characterization, HPLC analyses, mass spectra, and amino acid analyses appear in a data supplement available with the online version of this article (http://www.clinchem.org/content/vol49/issue1/). A description of small-scale purification of hCG and related molecules has appeared earlier (16), as has a progress report on the project (17). Evaluation of the final ampouled standards, including stability studies, value assignment, and comparisons of immunoassay results, will be the subject of separate reports.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
antibodies for western blots
Monoclonal antibodies (mAbs; IDC) with selected specificities were chosen for Western blots: INN(sbruck)-hCG-22 (recognizes hCG and hCGß-variants; broad specificity; epitope ß2, located on the top of ß-strand loops 1 and 3 of hCGß; amino acid regions hCGß20–25 and hCGß68–77); INN-hFSH-100 (hCG and hCG; broad specificity; epitope 2 on ß-strand loop 1 of hCG; amino acid region hCG13–22), INN-hCG-80 (hCG-specific; no recognition of dimer; epitope 6 in the region of the subunit interaction site, amino acids hCG33–42), and ISOBM-313, directed against amino acids hCGß141–144 (epitope ß8) (13)(18)(19)(20).
analytical procedures
The methods for amino acid analysis, amino acid sequence analysis, and RP-HPLC were described previously (16). Carbohydrate analyses were performed by the NIH-funded Research Center for Biomedical Complex Carbohydrates (Athens, GA). Monosaccharide composition was analyzed by gas chromatography–mass spectrometry on a HP 5890 gas chromatograph equipped with a Supelco DB1 fused-silica capillary column and interfaced to a 5970 mass spectrum detector. Trimethylsilyl derivatives of the methyl glycosides were prepared from 80 µg of each sample by methanolysis in 1 mol/L HCl in methanol at 80 °C (18–22 h), followed by re-N-acetylation with pyridine and acetic anhydride (for detection of amino sugars). The samples were then treated with Tri-Sil (Pierce Chemical)
sodium dodecyl sulfate–polyacrylamide gel electrophoresis, isoelectric focusing, and western blotting
The purity and composition of the various preparations were assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (13)(21). Briefly, proteins were loaded on precast polyacrylamide gradient gels (4–20%; Novex) at equimolar concentrations and separated by SDS-PAGE (Mighty Small II; Hoefer Scientific Instruments) at 200 V for 50 min under nonreducing conditions. For isoelectric focusing (IEF), dry acrylamide gels (6% T) were reswollen to their original thickness in 3% ampholyte solution (Ampholine 3.5–9.5; Amersham Biosciences) with or without the addition of 9 mol/L urea. Protein solutions were diluted with 1 g/L bovine serum albumin in distilled water, and 5-µL aliquots were loaded (300 V, 5 mA, 5 W for 30 min) and separated (3000 V, 5 mA, 5 W for 120 min) at 20 °C.
Proteins were electrophoretically transferred from SDS-PAGE (3.5 h, 400 mA; Trans Blot® Cell; Bio-Rad) or IEF gels (60 min) onto PVDF membranes (Immobiline®; Millipore) by semidry Western blotting (22). Membranes were blocked for 30 min in Tris-buffered saline-Tween buffer (0.02 mol/L Tris-HCl, 0.15 mol/L NaCl, 20 mL/L Tween 20, 0.1 g/L sodium azide, 30 g/L skim milk powder) and then incubated overnight in appropriate dilutions of reference mAbs [diluted in phosphate-buffered saline-Tween (0.05 mol/L sodium phosphate, 0.15 mol/L NaCl, 20 mL/L Tween 20, pH 7)] (13)(18)(20). After extensive washing with phosphate-buffered saline, membranes were incubated for 1 h with goat anti-mouse IgG-horseradish peroxidase (Immunopure; Pierce) diluted 1:1000 in 10 g/L milk powder in phosphate-buffered saline (0.15 mol/L NaCl, 20 mmol/L sodium phosphate, pH 7.4). Chemiluminescent substrate conversion (Super Signal West Dura; Pierce) was detected with Hyperfilm ECL (Amersham International).
time-resolved immunofluorometric assays
The coating, blocking, and incubation steps of the immunofluorometric assays (IFMAs) for hCG, hCGß, hCGßcf, and hCG were performed as described for human follicle-stimulating hormone (hFSH) (23). The characteristics of the mAbs against hCG, hCGß, hCGßcf, hCG, and hFSH have been described (13)(18). These mAbs (IDC) served as RRs in the international TD-7 Workshop on antibodies to hCG and hCG-related molecules (13). The coating mAbs were coded INN(sbruck)-45 (hCG+hCGn assay), -68 (hCGß assay), -106 (hCGßcf assay), and INN-hCG-72 (hCG assay). The detection mAbs (INN-hFSH-158 for the hCG-assay; INN-hCG-22, a pan-ß mAb), for all four other assays (18) were labeled with isothiocyanatophenylene triaminotetraacetic acid-europium (Wallac) according to the manufacturer’s recommendations. Hormone standards (hCG 3rd IS 75/537, hCGß 1st IRP 75/551, hCG 1st IRP 75/569) used to determine the sensitivities and specificities of the IFMAs for hCG, hCGß, and hCG were kindly provided by the National Institute for Biological Standards and Control. hCGßcf was a gift by Drs. Klaus Mann and Rudy Hoermann (University of Essen, Essen, Germany).
Inter- and intraassay CVs for each IFMA were <10% over the entire assay ranges (18). The new RRs were diluted in 0.01 mol/L NaHCO3 containing 1 g/L bovine serum albumin, aliquoted, snap-frozen in liquid nitrogen, and stored at -80 °C until assayed.
summary of purification protocols
Flow charts for the purification procedures are shown in Fig. 2
(hCG, hCGn, and hCGßcf) and Fig. 3
(hCG, hCGß, and hCGßn). Details of the purification procedures appear in the online data supplement (http://www.clinchem.org/content/vol49/issue1/).
|
|
biological activity
The biological activities of the hCG preparations were estimated in vivo in male rats by the seminal vesicle weight method (24) and in vitro by the Leydig cell method with testosterone production as the endpoint (25)(26). The 3rd IS (75/537) was used as a standard. The in vivo assays were performed in two laboratories and the in vitro determinations in one.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
10% as much hCGßcf as that in lots of 10 or 20 years earlier. The method developed in 1972 and used to produce the 3rd IS [CR119; Canfield–Ross preparations of hCG made of Columbia University (CR)-quality hCG] (2) was improved by use of modern chromatographic media and the addition of hydrophobic interaction chromatography (Fig. 2
). This permitted separation of hCGn and other minor contaminants from hCG. Some free fragments of hCGßCTP (ß117–145 and other similar fragments) were also separated by this step. It is not clear whether these peptides had not been removed by previous steps or whether they were introduced by cleavage by some contaminating protease. After gel filtration, hCGßcf was further purified with use of Blue Sepharose and RP-HPLC.
purification of subunits
Subunit purification was accomplished starting with the CR-quality hCG preparation (Fig. 3
). Although RP-HPLC can dissociate and separate subunits in a single step, oxidized forms and metabolites cause cross-contamination. Thus, oxidized forms of hCGß coeluted with hCG and oxidized forms of hCG with hCGßn. Consequently, the subunits were first separated by anion-exchange chromatography (2) and were further purified by RP-HPLC in 0.2 mL/L trifluoroacetic acid. The fractions were immediately brought to a higher pH, lyophilized, incubated to permit recombination with any complementary subunit, and gel-filtered to remove any reformed hCG as well as to reduce salt content. Exposure to low pH was minimized to reduce loss of sialic acid residues.
hCGßn was prepared directly from the RP-HPLC purification step used to prepare hCGß. An adequate quantity of hCGßn was isolated as a byproduct of the purification of hCGß. A small quantity of urinary ribonuclease remained in the hCGßn preparation and was not easily removed. Other investigators have also reported copurification of this protein with hCGßcf (27).
characterization of the preparations
The purity of each preparation was assessed by SDS-PAGE under reducing conditions (Fig. 4
), immunoblotting (Figs. 5
and 6
), IFMAs (Table 1
), and by N-terminal sequence analysis (Table 2
). The protein content of each preparation was quantified by amino acid analysis [Table S1 in the online data supplement (http://www.clinchem.org/content/vol49/issue1/)]. Comparison of the known primary structure with the calculated amino acid content showed good agreement.
|
|
|
|
|
SDS-PAGE (Fig. 4
) confirmed the expected band patterns of purified hCG, hCGß, and hCG as well as the two bands expected for hCGßcf. Analyses of hCGn by RP-HPLC indicated the presence of 10–15% of nonnicked hCG.
N-Terminal sequence analyses indicated that each preparation contained <2–3% contaminants; the limit of detection of the method (Table 2
). The single exception was the hCGßn preparation, which was contaminated with some urinary ribonuclease (27) as described above.
Characterization of the preparations by IEF and subsequent Western blotting (Figs. 5
and 6
) with pan anti--subunit and pan anti-ß-subunit antibodies showed that hCG consists of three major bands with an approximately neutral pI. The various forms of hCG- and hCGß-derived variants displayed at least six major bands around a pI of 4.5. hCGn and hCGßn appear to be more acidic than hCG and hCGß. hCGßcf, with a pI >9.5, could not be analyzed by this approach. When hCG and hCGn were analyzed by IEF in 9 mol/L urea, the expected subunit patterns were observed (Fig. 6
).
The specific absorbance values for the preparations of hCG and its subunits differed slightly from published values, which were also based on protein content determined by amino acid analysis (Table 3
) (2)(16)(28). The extinction values for the 3rd IS were determined by absorbance at 280 nm, whereas for the current preparation, 276 nm was used, possibly contributing to this difference. However, the greater purity of the new preparation could also be responsible for the observed difference.
|
Results of carbohydrate analysis, RP-HPLC profiles, and mass spectra of the preparations are given in the online data supplement. The mean molecular weights were as follows: hCG, 37 500; hCGß, 23 500; hCG, 14 000; and hCGßcf, 10 500. Carbohydrate composition [Table S2 in the online data supplement (http://www.clinchem.org/content/vol49/issue1/)] agreed reasonably well with the expected content of each of the sugars. The sialic acid content (mol/mol) and the expected ranges were as follows: 9.8 (12–16) for hCG; 2.0 (2–4) for hCG; 13.3 (8–12) for hCGß; 8.5 (12–16) for hCGn, and 8.8 (8–12) for hCGßn. The reason for the higher than expected sialic acid content for hCGß is not known and may be an experimental artifact. Complete compositional carbohydrate analysis was not performed for CR119 hCG, the 3rd IS, but the sialic acid content of CR119 hCG (10.6% by weight) was very similar to that of the new preparation (11.1% by weight). The percentage of carbohydrate by weight compared with protein weight for the purified lyophilized proteins was as follows: hCG, 29%; hCG, 29%; hCGß, 43%; hCGn, 35%; hCGn, 35%; hCGßn, 83%; and hCGßcf, 14%. The reason for the high sugar content of hCGßn is not known. This is obviously not protein-bound sugar but other sugar material mixed with the purified protein. Some glucose was also found, perhaps originating from the dextran columns used during purification.
biological activity
The median in vivo biopotency (with the 95% confidence intervals) measured by one laboratory was 15 400 (14 200–16 800) IU/mg, whereas in the other laboratory, it was 10 300 (8360–12 600) IU/mg. This laboratory also determined the in vitro biological potency to be 14 100 IU/mg (12 500–16 000).
properties of the new 1ST rr for immunoassay of HCG
The improved overall properties of the new hCG preparation 99/688 are compared with those of the 3rd/4th IS in Table 4
. The free subunits were also improved to the same extent in terms of peptide bond intactness and freedom from contaminants, as described in the analysis above.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Preparation of the new RRs was achieved by use of an initial purification scheme similar to that for preparation of the 3rd IS of hCG, but more modern chromatographic media provided better separation, although care was taken to retain the microheterogeneity of hCG and its subunits, as revealed by IEF. This microheterogeneity primarily reflects the differences in sialic acid content typical of all glycoprotein hormones, and retention of these natural variants in the final preparations appears desirable. Removal of contaminating forms of hCG and other proteins was achieved by addition of hydrophobic interaction chromatography and RP-HPLC to the purification scheme. Use of hydrophobic interaction chromatography also facilitated removal of any residual traces of protease activities, such as those present in the 3rd IS (3)(12)(29) as well as in hCGn and hCGßcf (15)(16).
Preparation of subunits of superior quality to the last subunit standards, IRP 75/569 and IRP 75/551, was difficult. Direct use of RP-HPLC to separate subunits in a single step was not feasible because oxidized subunits coeluted with the other subunit or with the metabolites. Methionine residues are easily oxidized, and when the single methionine in the hCGß subunit is oxidized, hCGß coelutes with hCG. Because hCG contains three methionines, it may elute as four different peaks. Anion-exchange chromatography in urea buffer did not separate oxidized from nonoxidized subunits but did allow good separation of each of the subunits. RP-HPLC was used subsequently to remove oxidized forms from each subunit. Further removal of any complementary subunit was achieved by allowing subunit recombination followed by gel filtration.
Our procedures relied heavily on analytical screening of every column fraction by RP-HPLC to decide which fraction should become part of the pool at each step. The large-scale procedure adopted facilitated production of the large quantity of protein required for IRPs. The preparations were thoroughly characterized with respect to protein and carbohydrate content and the presence of immunoreactive cross-contaminants. Amino acid sequence analysis was essential to verify polypeptide chain integrity and the extent of peptide bond cleavages in nicked materials. Because the sensitivity of Edman sequence analysis for detection of contaminating proteins is 3%, highly sensitive IFMAs and SDS-gel electrophoresis in conjunction with Western blotting were used for more sensitive assessment of purity.
Compared with the 3rd/4th IS for hCG, the new 1st RR for hCG contains much less hCGn, hCGß, hCG, and hCGßcf (Table 1
). Estimation of the content of contaminating forms of hCG by time-resolved IFMAs showed that the preparations of hCG, hCGß, hCGß, and hCG were of excellent purity. Only hCGn contained substantial amounts (>1%) of hCG, (-CTP)hCGß(n), and hCG (Table 1
and Figs. 4
and 5
). These results were confirmed by Western blotting (Fig. 6
). No hCG-related contaminants were observed in hCGß, hCGßcf, and hCG. There are minute amounts of (a) -CTPhCG (hCG lacking the C-terminal portion of hCGß) and hCG in hCG 99/688, (b) -CTPhCGß(n) in hCGßn, and (c) -CTPhCG and -CTPhCGß(n) and hCG in hCGn. Presumably, a somewhat larger amount of hCGß/ßn can be found in hCGn. Because the mAbs used for Western blotting recognize the various forms of hCG/hCGß with different affinities, the method is not quantitative. Nevertheless, it provides a qualitative screen for those hCG-derived variants for which no purified preparations are available, i.e., hCG and hCGß variants truncated at their COOH terminus (-CTPhCG, -CTPhCGn, -CTPhCGß, and -CTPhCGßn).
The combination of all these methods facilitated accurate characterization of the preparations and demonstration of their very high quality. Two metabolite preparations were somewhat contaminated: hCGn contained 10% hCG, whereas hCGßn contained 10% urinary ribonuclease and was the only preparation with detectable foreign protein. This does not prohibit their use for characterization of the specificity of immunoassays for hCG and its subunits.
The sialic acid content of the new reference preparation of hCG is similar to that of CR 119 (the 3rd IS). The major change in protein content observed by N-terminal sequence analysis was complete removal of the peptide bond cleavages within the ß-subunit at positions 44–45 and 47–48 (9). This, together with the IEF patterns observed (Fig. 6
) and the excellent in vivo and in vitro biological activity demonstrated, provides assurance that the hydrophobic step selected molecular species representative of the physiologically relevant active hormone.
The availability of the new reference preparations provides a sound scientific basis for improving the standardization of assays for hCG-related molecules. The 3rd IS for hCG (75/537) was calibrated by bioassay in international units (IU) against the 2nd IS. The subunits, which lack bioactivity, were assigned arbitrary units (also IU) based on mass. This common unitage falsely suggests that such "standardization" of hCG and its subunits has a common basis. Value assignment on the basis of biological activity is appropriate for hCG standards intended for therapeutic purposes. In contrast, for hCG standards used to calibrate immunoassays, value assignment in substance concentrations on the basis of amino acid analysis, as for the RRs reported here, is much more informative. As an important first step toward improving the comparability of immunoassays for hCG, the new standards will allow manufacturers to calculate the cross-reactivity of these six hCG-related molecules in the immunoassays they supply. Inclusion of such data in assay inserts will enable direct comparison of the relative recognition of these molecules in different assay systems. This should be achievable for currently available hCG immunoassays.
Realistically, the new standards are likely to be used for primary calibration of the next generation of hCG immunoassays rather than for current assays. Careful consideration will then be required as to how best to transfer values for the primary (nonnicked) hCG standard to the "working" secondary standards used by manufacturers, because the latter are likely to contain substantial amounts of nicked hCG. At the same time, convincing clinical colleagues of the advantages of adopting molar units for hCG will present a major challenge to laboratorians.
The use of molar units is particularly helpful when the ratios of various forms of hCG are compared, as in the diagnosis of Down syndrome and trophoblastic tumors. Some groups already report results for hCG, its subunits, and fragments in molar concentrations based either on locally prepared standards or on theoretical values calculated from the WHO standards (30)(31). Value assignment in substance concentrations also increases the probability that subsequent standards can be calibrated identically. [Unfortunately this is not possible retrospectively for the 3rd/4th IS because these preparations contain carrier protein.] Measurement of the molar content of protein by amino acid analysis also eliminates some of the practical problems associated with glycoproteins, e.g., difficulties in gravimetric determination of glycoprotein mass because of tight binding of water molecules. Although carbohydrate residues present on glycoproteins rarely affect immunoreactivity, they may strongly affect biological activity, e.g., by modulating the half-lives of circulating glycoproteins or by influencing receptor binding and activation. Because antibodies almost exclusively recognize peptide epitopes, the molar content of proteins, rather than their bioactivity, may be expected to best reflect what is measured by immunoassays.
Although antibodies that recognize glycan-associated epitopes within hCG are very rare (32), the potential utility of measurement of hyperglycosylated hCG (acid variants of hCG with high carbohydrate content) in early pregnancy and in cancer patients has been the subject of recent reports (33)(34). The current interest in hyperglycosylated hCG is related to the hCG isoforms recognized by antibody B152, which is directed to the glycans and peptide structure within the hCGß COOH-terminal peptide (CTP) region (35), but this antibody does not recognize all forms of hyperglycosylated hCG., e.g., those with modifications in the N-glycan region only. Preparation of a reference standard for hyperglycosylated hCG would be desirable but was not part of the focus of the current project.
It is not likely, however, that the presence of various hyperglycosylated hCG forms in serum or urine causes errors in current commercial assays for hCG. It has recently been shown by IEF, Western blotting, and sandwich immunoassays that all mAbs tested, including those against all major epitopes located in the immunodominant regions such as the top of the two adjacent ß-strand loops 1 and 3 of hCGß, recognize the entire spectrum of glycosylation variants, including deglycosylated hCG, asialo-hCG, variants of more neutral pI, and highly acidic (hyperglycosylated) variants of pregnancy hCG as well as highly purified acidic hCG variants of tumor patients (21). All of these epitopes are therefore predominantly determined by the protein backbone, and the carbohydrate moieties that form a biochemical basis for hCG heterogeneity are neither of major antigenic relevance nor are they structurally related to any immunodominant antigenic region. Most manufacturers of "hCG" reagent sets use mAbs with epitope specificities identical to the reference mAbs used for these studies (13). The calibration of the RRs in substance concentrations (mol/L) is undoubtedly an important advantage for the measurement of hCG that is heterogeneous in nature with respect to glycosylation.
In conclusion, we have produced highly purified preparations of hCG, hCG, hCGß, and three partially degraded forms of these. The protein contents of these preparations were determined by amino acid analysis. The WHO has approved these preparations as RRs, and their use should facilitate improved comparability among immunoassays for measurements of hCG, its subunits, and its metabolites. The characterization of the final WHO preparations as well as their application in external quality assessment (proficiency testing) schemes will be reported separately.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
and hCGß, and ß subunits of hCG; hCGßcf, ß-core fragment of hCGß; hCGn, nicked hCG; hCGßn, nicked hCGß; RR, Reference Reagent; mAb, monoclonal antibody; RP-HPLC; reversed-phase HPLC; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; IEF, isoelectric focusing; IFMA, immunofluorometric assay; hFSH, human follicle-stimulating hormone; CR, Canfield–Ross preparation; and CTP, hCGß COOH-terminal peptide. 
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ß subunits of human chorionic gonadotrophin for immunoassay. J Endocrinol 1980;84:295-310.[ISI][Medline]
[Order article via Infotrieve]
-subunit. J Endocrinol 1988;117:147-152.[Abstract]
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  Z. Cao and R. Rej Are Laboratories Reporting Serum Quantitative hCG Results Correctly? Clin. Chem., April 1, 2008; 54(4): 761 - 764. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Casella, H. Lindner, C. Zenzmaier, D. Riitano, P. Berger, and T. Costa Non-Gonadotropin-Releasing Hormone-Mediated Transcription and Secretion of Large Human Glycoprotein Hormone {alpha}-Subunit in Human Embryonic Kidney-293 Cells Endocrinology, March 1, 2008; 149(3): 1144 - 1154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. Sigel and D. G. Grenache Detection of Unexpected Isoforms of Human Chorionic Gonadotropin by Qualitative Tests Clin. Chem., May 1, 2007; 53(5): 989 - 990. [Full Text] [PDF]
|
|
|
|
 |
|
 U.-H. Stenman, A. Tiitinen, H. Alfthan, and L. Valmu The classification, functions and clinical use of different isoforms of HCG Hum. Reprod. Update, November 1, 2006; 12(6): 769 - 784. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Handelsman The Rationale for Banning Human Chorionic Gonadotropin and Estrogen Blockers in Sport J. Clin. Endocrinol. Metab., May 1, 2006; 91(5): 1646 - 1653. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Bunk and M. J. Welch Characterization of a New Certified Reference Material for Human Cardiac Troponin I Clin. Chem., February 1, 2006; 52(2): 212 - 219. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bristow, P. Berger, J.-M. Bidart, S. Birken, R. Norman, U.-H. Stenman, C. Sturgeon, and on behalf of the IFCC Working Group on hCG Establishment, Value Assignment, and Characterization of New WHO Reference Reagents for Six Molecular Forms of Human Chorionic Gonadotropin Clin. Chem., January 1, 2005; 51(1): 177 - 182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. von Lode, J. Rainaho, and K. Pettersson Quantitative, Wide-Range, 5-Minute Point-of-Care Immunoassay for Total Human Chorionic Gonadotropin in Whole Blood Clin. Chem., June 1, 2004; 50(6): 1026 - 1035. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U.-H. Stenman Standardization of Assays for Human Chorionic Gonadotropin Clin. Chem., May 1, 2004; 50(5): 798 - 800. [Full Text] [PDF]
|
|
|
|
|
|
L. A. Cole, J. M. Sutton, T. N. Higgins, and G. S. Cembrowski Between-Method Variation in Human Chorionic Gonadotropin Test Results Clin. Chem., May 1, 2004; 50(5): 874 - 882. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-L. Frendo, J. Guibourdenche, G. Pidoux, M. Vidaud, D. Luton, Y. Giovangrandi, D. Porquet, F. Muller, and D. Evain-Brion Trophoblast Production of a Weakly Bioactive Human Chorionic Gonadotropin in Trisomy 21-Affected Pregnancy J. Clin. Endocrinol. Metab., February 1, 2004; 89(2): 727 - 732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. T.A. Borrelli, S. A. Butler, S. M. Docherty, E. M. Staite, A. L. Borrelli, and R. K. Iles Human Chorionic Gonadotropin Isoforms in the Diagnosis of Ectopic Pregnancy Clin. Chem., December 1, 2003; 49(12): 2045 - 2049. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U.-H. Stenman Standardization as a Private Enterprise Clin. Chem., April 1, 2003; 49(4): 535 - 536. [Full Text] [PDF]
|
|
| ||||||
