Steven Birken

The ISOBM TD-7 Workshop on hCG and Related Molecules

The ISOBM TD-7 hCG Workshop was established to characterize the molecular epitope structure and specificities of a panel of diagnostically relevant monoclonal antibodies (MAbs) directed against human chorionic gonadotropin (hCG) and its derivatives, and to consider how this information could be used to improve comparability of immunoassay results for these analytes. In this multicenter study, 27 MAbs have been characterized in detail as to their main and fine specificities by direct binding-, competitive- and sandwich-RIA, -ELISA, BIAcore® and Western blotting. Antigens used in the study included the upcoming first WHO reference reagents for immunoassay, i.e. nick-free hCG (hCG), nicked hCG (hCGn), hCG α-subunit (hCGα), hCG β-subunit (hCGβ), nicked hCG β-subunit (hCGβn), hCG β-core fragment (hCGβcf), synthetic peptides of hCGβ C-terminal peptide (hCGβCTP), and homologous hormones, luteinizing hormone (LH) and subunits (LHβ) from various species. Correct classification of blinded internal controls demonstrated the reliability of the MAb referencing approach. Three-dimensional molecular epitope assignment was possible in many instances by comparing immunoreactivity of the ISOBM MAbs (n = 27) to a large panel of MAbs (n = 18) previously well characterized in the Innsbruck (P.B.) and Paris (J.M.B.) laboratories. All three major antibody specificities (α, n = 1; β, n = 21; αβ, n = 5) were represented in the TD-7 MAb panel. HCGβ MAbs could further be subdivided into (i) those recognizing hCGβ only (epitopes: β6, n = 1; β7, n = 2; β14, n = 1) and (ii) those recognizing hCGβ + hCG (β1, β2, β4, β5, n = 10; β8 and β9, n = 9). Members of the latter group were specific either for hCG + hCGβ + hCGβcf (β1, n = 3) or hCG + hCGβ + hCGβCTP (β8, n = 6; β9, n = 1) or in addition to hCG + hCGβ + hCGβcf recognized hLH/hLHβ to a minor (β2, n = 3; β4, n = 3) or similar degree (β5, n = 1). Epitopes were (i) located on the first and third loops protruding from the cystine knot of hCGβ (β2–β6, aa hCGβ20–25 and 68–77), (ii) presumably centered around the knot itself (β1), or (iii) on hCGβCTP (epitope β8 = hCGβ141–144, β9 = hCGβ113–116). The ISOBM panel of MAbs represents all major epitope specificities suitable for the design of specific sandwich immunoassays. High analyte variability in serum and urine during the course of pregnancy and tumor development favors certain epitope combinations. For routine diagnostic purposes, assays recognizing a broad spectrum of hCG/hCGβ variants such as hCG + hCGn + hCGβ + hCGβn + hCGβcf + –CTPhCG + –CTPhCGβ may be useful. Low cross-reactivity against related glycoprotein hormones (e.g. hLH) and their derivatives is mandatory. These criteria are best met by combinations of MAbs directed against epitopes located around the cystine knot (β1) and against those encompassing the top of loops 1 and 3 on hCGβ (β2, β4). The first WHO reference reagents for immunoassay of hCG and hCG-related molecules being prepared by the IFCC should facilitate characterization of what assays for ‘hCG’ are measuring. The next step towards improving between-laboratory comparability of measurements of hCG/hCG derivatives in pregnancy and oncology is provided by results of this TD-7 Workshop.

Differences in Recognition of the 1st WHO International Reference Reagents for hCG-Related Isoforms by Diagnostic Immunoassays for Human Chorionic Gonadotropin

BACKGROUND The 1st WHO International Reference Reagents (IRRs) for 6 human chorionic gonadotropin (hCG)-related molecular variants, highly purified and calibrated in substance concentrations by the IFCC Working Group for hCG, permit experimental elucidation of what commercially available hCG methods measure in molar terms and enable assessment of their fitness for clinical purposes. METHODS Pools containing known amounts of the IRRs spiked into normal human serum were issued to participants through the UK National External Quality Assessment Service for hCG for a period of 7 years. Among 16 assays used, 4 recognized only hCG, whereas 6 recognized hCG and its free beta-subunit (hCGbeta), and 6 recognized hCG, hCGbeta, and the beta core fragment. RESULTS Differences in calibration of current hCG assays are moderate. Mean recovery of the current International Standard (IS), hCG IS 75/589, was 107% (range 93% to 126%), whereas that of the IRR 99/688 for hCG was 139% (range 109%-164%). Between-method variation for the latter (CV 12.3%) was also greater than for IS 75/589 (CV 8.8%). Recognition of hCGbeta varied markedly (CV 37%). Most assays overestimated it, but 2 RIAs produced results that were slight underestimations. Recognition of the beta core fragment was even more variable (CV 57%) and was closest to equimolarity for the RIAs. CONCLUSIONS Assays for hCG show considerable variation in their recognition of various forms of hCG, and this variability is the most important cause of method-related differences in hCG results in serum and an even more important cause of method-related differences in urine measurements. Equimolar recognition of the major hCG isoforms is essential if between-method comparability for hCG is to be improved.

Development and characterization of antibodies to a nicked and hyperglycosylated form of hCG from a choriocarcinoma patient: generation of antibodies that differentiate between pregnancy hCG and choriocarcinoma hCG.

Human chorionic gonadotropin (hCG) exists in blood and urine as a variety of isoforms one of which contains peptide bond cleavages within its β-subunit loop 2 and is referred to as nicked hCG (hCGn). This hCG isoform appears to be more prevalent in the urine of patients with certain malignancies and possibly in some disorders of pregnancy. Until now, only indirect immunoassays could be used to quantify hCGn. We report the development of two monoclonal antibodies (MAbs) to a form of hCGn isolated from a choriocarcinoma patient. This hCG isoform was not only 100% nicked, but also contained 100% tetrasaccharide-core O-linked carbohydrate moieties in its β COOH-terminal region. Two-site immunometric assays have been developedusing these new antibodies, B151 and B152. The former exhibits good specificity for hCGn independent of the source of the hCGn, the form excreted by choriocarcinoma patients or the form of hCGn from normal pregnancies. The latter antibody, B152, is sensitive to the carbohydrate moieties and possibly other differences in hCG isoforms, but is not for nicking of the β-subunit. These two immunometric assays provide potential novel diagnostic tools for direct measurement of hCG isoforms which could not be accurately quantified earlier before development of the assays using these newly generated antibodies.

An oligosaccharide of the O-linked type distinguishes the free from the combined form of hCG [alpha] subunit

JAR malignant trophoblast cells produce a free alpha subunit in addition to an alpha combined with beta subunit as hCG. The free alpha is larger by gel chromatography and SDS-PAGE than combined alpha and is unable to associate with beta subunit to form hCG. A tryptic fragment, representing amino acid residues 36-42, derived from free alpha was larger than the corresponding fragment from combined alpha. After neuraminidase treatment, the fragment from free alpha bound peanut lectin agarose, which is specific for Gal beta 1-3GalNAc as found in O-linked oligosaccharides. The fragment also contained Gal and GalNAc (and a lesser amount of GlcNAc) as determined by glycosidase sensitivity and amino sugar analyses. Removal of this tryptic fragment ablated the size difference between free and combined alpha subunits.

The structures of the serine-linked sugar chains on human chorionic gonadotropin

The human chorionic gonadotropin beta-subunit tryptic COOH-terminal peptide (residues 123-145) which contains 3 serine-linked sugar chains was isolated. The sugar chains were cleaved by beta-elimination and then separated by gel filtration. The peaks were pooled and their compositions determined. The products of serial glycosidase digestion and periodate oxidation of the intact glycopeptide were also characterized. Of the serine-linked sugar chains, 13% were the hexasaccharide NeuAc alpha 2,3 Gal beta 1,3 (NeuAc alpha 2,3 Gal beta 1,4 GlcNAc beta 1,6) GalNAc, 34% the tetrasaccharide NeuAc alpha 2,3 Gal beta 1,3 (NeuAc alpha 2,6) GalNAc, 43% the trisaccharide NeuAc alpha 2,3 Gal beta 1,3 GalNAc and 10% the disaccharide NeuAc alpha 2,6 GalNAc.

Partial amino acid sequence of human placental lactogen precursor and its mature hormone form produced by membrane-associated enzyme activity

Abstract The precursor form of human placental lactogen, synthesized by a wheat germ extract cell-free system, has been partially sequenced and found to contain a high percentage of leucine residues within its first 20 amino acids. The partial NH 2 -terminal structure appears to be: The precursor form of hPL, produced by an ascites extract cell-free system, was cleaved by a membrane-associated enzyme into a form which exhibits the methionine and valine residues in NH 2 -terminal positions identical to those of native human placental lactogen.

Metabolism of hCG and hLH to multiple urinary forms.

Human chorionic gonadotropin (hCG) is synthesized primarily in the placenta while human luteinizing hormone (hLH) is produced in the pituitary. Both hormones are highly homologous in structure and both appear to be altered to analogous molecular forms as the hormones are proteolytically processed, or metabolized, from tissue of origin, through the circulation, and finally to the urine. Placental hCG is excreted into urine as heterodimeric hormone, heterodimeric nicked hCG, free subunits (some nicked), and predominantly as the hCG beta core fragment. A pituitary form of heterodimeric hCG, which is partly sulfated as is pituitary hLH, was recently isolated and is likely the form of hCG observed in the urine of healthy postmenopausal women and nonpregnant premenopausal women as well. A pituitary form of the hLH beta core fragment, highly analogous in structure to that of urinary hCG beta core fragment, has been used to develop specific monoclonal antibody assays to measure urinary hLH beta core fragment which is excreted at significantly higher molar concentrations than is hLH in the urine of ovulating women 1 or 2 days after the LH surge. This fragment of LH appears in the urine of postmenopausal women as well. The development of the capability to distinguish the hCG beta core fragment from the hLH beta core fragment in urine may have useful applications in tumor marker assays, pregnancy tests, and menopause. While hCG urinary assays have been widely employed, urinary assays for hCG and hLH metabolites are much less used since the urinary molecular forms are only partly known. Our studies of hCG and hLH urinary metabolites are directed towards improvement of the utility of urinary measurements of molecules derived from these hormones. Since many of the molecular forms of these two hormones in urine differ from their forms in blood, it may be necessary to produce new immunoassays as well as novel urinary reference preparations to accurately measure these molecules within their urinary matrix.

Immunochemical Measurement of Early Pregnancy Isoforms of hCG: Potential Applications to Fertility Research, Prenatal Diagnosis, and Cancer

Human chorionic gonadotropin, the glycoprotein hormone of pregnancy, is found naturally in blood and urine in a variety of isoforms. These variants are related to both peptide bond cleavages (such as the nicked forms of hCG) and the beta core fragment urinary metabolite, as well as the larger variety of species resulting from carbohydrate heterogeneity. We have recently developed immunoassay systems that can measure nicked forms of hCG (antibody B151) as well as particular high carbohydrate variants (hyperglycosylated forms) of hCG (B152), which are associated with cancers producing hCG. Using the assay system for nicked hCG, we found that nicked hCG does not appear to be present as a significant hCG isoform during normal pregnancies if the urine specimens are well preserved. Applying the assay for hyperglycosylated hCG isoforms, we discovered that these forms are prevalent during very early pregnancy and decline rapidly to low concentration after the first 6 weeks of pregnancy. Persistence of these early pregnancy forms does not bode well for the pregnancy. Other investigators report that measurement of such hCG isoforms may aid in diagnosis of Down syndrome pregnancies. In summary, measurement of the hyperglycosylated hCG isoforms are useful for evaluation of healthy progress of normal pregnancy, as an additional detection marker for Down syndrome pregnancies, and as a potential new marker of trophoblastic malignancy. New reference preparations will soon be available for the calibration of assay systems for measurement of many of these hCG variants and metabolites.

Standardization Of Protein Immunoprocedures Choriogonadotropin (Cg)

The objectives of this report is to improve the quality of immunochemical determinations of proteins. Procedures for determination of the various molecular forms hCG were chosen as the subject of a detailed study, with the aim as using this report as a model for similar future projects. The aim of this undertaking is to improve the reliability and reproducibility of the results and, as a result of this, to enhance the clinical usefulness of hCG determinations. The following means will be used to achieve these goals: (a) establishing uniform nomenclature and abbreviations for various molecular forms of hCG, (b) preparation of new calibrators for these and establishing of methods for determining the amount of substance concentration in mol/L, (c) improving quality assessment materials and procedures, (d) definition of methods for characterization of hCG measurement procedures.

Expression of human chorionic gonadotropin uniformly labeled with NMR isotpes in Chinese hamster ovary cells: An advance toward rapid determination of glycoprotein structures

SummaryMost secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure requires confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein back bone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with 13C and 15N isotopes. We report the first such uniform labeling of a glycoprotein using a universal 13C-and 15N-labeling medium to express 13C, 15N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The 13C, 15N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.