Characterization of a carboxyterminal peptide fragment of the human choriogonadotropin beta-subunit excreted in the urine of a woman with choriocarcinoma.

Amr, Sania; Wehmann, Robert E.; Birken, Steven; Canfield, Robert E.; Nisula, Bruce C.

doi:10.1172/jci110773

Cited by 36 publications

(11 citation statements)

References 40 publications

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“…Standardizing assays that measure heterogeneous mixtures is difficult, and is probably impossible on theoretical grounds [56,57]. The only fully satisfactory solution will be to determine the principal components of such mixtures [58].…”

Section: Discussionmentioning

confidence: 99%

The ISOBM TD-7 Workshop on hCG and Related Molecules

et al. 2002

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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 encompassi...

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Section: Discussionmentioning

confidence: 99%

The ISOBM TD-7 Workshop on hCG and Related Molecules

et al. 2002

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“…Because such fragments were not usually found in the sera, they have been considered to be urinary products. Amr et al (5,23) have shown that carboxy-terminal peptide fragments of the hCGjfl were commonly contained in the urine of patients with gestational trophoblastic neoplasia but rarely in the urine of pregnant women and suggested that it could be accounted for by the metabolism of the forms of hCG that bear an altered carbohydrate structure. In this regard, interestingly, we have already found that all the c-hCG samples, including the same samples studied here, contain the unusual multiantennary complex-type sugar chains which are not found in normal pregnancy hCG (6).…”

Section: Discussionmentioning

confidence: 99%

“…Most of these works suggested that the carbohydrate structures of hCG change by neoplastic transformation of trophoblast (1)(2)(3). On the other hand, several investigators have reported the presence of low molecular weight hCG/3-related fragments in urine of patients with trophoblastic neoplasms (4,5). We found that definite structural changes commonly occurred in the sugar chains of choriocarcinoma urine hCG samples (6), and also observed that the same samples bearing such unusual carbohydrates released an unusual fragment, in addition to aand 0-subunits, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions with dithiothreitol (DTT) (7).…”

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Fragmentation of the β-Subunit of Human Chorionic Gonadotropin Produced by Choriocarcinoma*

et al. 1988

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When human chorionic gonadotropin (hCG) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions with dithiothreitol (DTT), a smaller weight material (CTP'), in addition to the beta-subunit, could be detected by Western blot analysis using antiserum for hCG beta-carboxy-terminal peptide (CTP). The CTP' band was much more apparent with urinary hCG from a patient with choriocarcinoma than with that from normal pregnant women. Second-dimensional electrophoresis of the choriocarcinoma hCG (c-hCG) after reduction with DTT indicated that the CTP', Mr 25,000, was released from the beta-subunit. The carbohydrate structure of the CTP' was analyzed by affinity with lectin-peroxidase on a nitrocellulose membrane. The CTP' did not interact with Concanavalin A, but exhibited strong interaction with both RCA120 and Arachis hypogaea after removal of sialic acid, indicating that it was released as a fragment containing an O-linked sugar chain as was found in the hCG beta carboxy-terminal portion. Western blot analysis using the antisera for hCG alpha, hCG beta, and hCG beta-CTP showed that the CTP' contains not only the carboxy-terminal portion but also a part of the internal (core) portion of the beta-subunit molecule. This dissociation of the c-hCG beta was further supported by the presence of a faster moving component (FMC) which may correspond to the NH2-terminal side counterpart. The desialylated FMC could be detected by Concanavalin A and RCA120 but not by Arachis hypogaea, indicating that it contains N-linked rather than O-linked sugar chains. The FMC does not contain any of the epitopes for the antisera examined in Western blot. These results indicate that the beta-subunit of the choriocarcinoma urine hCG has an unusual site which is dissociated into two components of Mr 25,000 (CTP') and Mr 18,000 (FMC) by DTT reduction.

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“…When the ratio of Ag-2 to Ag-1 was compared in urinary hCG preparations from various patients, it was found that the ratio was very low in normal pregnancy but increased in hydatidiform mole and was the highest in choriocarcinoma. In recent studies, Masure et al (1981) and Amr et al (1983) also showed the presence of small immunoreactive hCG molecules in urine from patients with hCG-secreting neo¬ plasmas and choriocarcinomas, respectively. The latter authors proved that the small hCG compo¬ nents were hCG-ß carboxy-terminal fragments.…”

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confidence: 92%

Affinity to lectin, biological and immunological characteristics of human chorionic gonadotropins from pregnant women and trophoblastic tumour patients

Koyama

Toda

Kuriyama

et al. 1986

Acta Endocrinologica

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To compare the affinity to lectin of human chorionic gonadotropins (hCG) from pregnant women and trophoblastic tumour patients, a small amount of urine was fractionated by a lentil lectin (LcH) affinity chromatography. The LcH-bound fractions were eluted with 2% mannoside solution, and each fraction was assayed for hCG activities by radioimmunoassay. In pregnant women, more than 90% of hCG immunoactivity in urine was bound to the LcH column and eluted from it, whereas 8 to 26% and 37 to 51% of the activity were not adsorbed to the affinity column and were recovered in the LcH-unbound fraction in the patients with hydatidiform mole and choriocarcinoma, respectively. These results suggest that LcH affinity chromatography of urinary hCG contributes to differential diagnosis between pregnancy and trophoblastic tumours. To characterize the properties of hCG from the urine of choriocarcinoma patients, with or without the LcH affinity, hCG activities in both LcH-unbound and LcH-bound fractions were measured by in vivo and in vitro bioassays and each of the activities was compared with the activities measured by radioimmunoassay. The results showed that the LcH-unbound fraction contained hCG molecules defecting to induce the biological activity of hCG in vivo.Human chorionic gonadotropin (hCG) is a glyco¬ protein hormone synthesized by placenta and trophoblastic tumours. It was interesting to study whether the hCG molecules secreted from differ¬ ent sources are the same or not. In earlier studies, Reisfeld et al. (1959) and Reisfeld & Hertz (1960) reported different physiochemical properties of hCG from normal pregnant women and patients with trophoblastic tumours. Wide & Hobson (1964, 1967) also showed different ratios between the biological and immunological activities of hCG secreted from normal pregnancy, hydatidiform mole and choriocarcinoma. However, Lewis et al. (1964) could not find any antigenic differences between the two gonadotropins from pregnancy and choriocarcinoma. Our previous studies (Iso¬ jima et al. 1969; Koyama 1971) indicated that crude urinary hCG preparations from patients with trophoblastic tumours contained two anti¬ genic components of hCG with different molecu¬ lar size which could be separated from each other by gel filtration on Sephadex G-100 column. The immunological and biological studies of these two components revealed that one large component (Ag-1) had both gonadotropic and immunologie activities of hCG, while the other small compo¬ nent (Ag-2) had only immunologie activity of hCG. By immunodiffusion test, Ag-1 cross-re¬ acted to Ag-2 with spar formation. These results were quite similar to those reported by Hamashige & Arquilla (1964) and Hamashige et al. ( 1966, 1967 who studied extensively the antigenic heterogeneity of hCG. When the ratio of Ag-2 to Ag-1 was compared in urinary hCG preparations from various patients, it was found that the ratio was very low in normal pregnancy but increased in hydatidiform mole and was the highest in choriocarcinoma. In recent studies, Mas...

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Characterization of a carboxyterminal peptide fragment of the human choriogonadotropin beta-subunit excreted in the urine of a woman with choriocarcinoma.

Cited by 36 publications

References 40 publications

The ISOBM TD-7 Workshop on hCG and Related Molecules

The ISOBM TD-7 Workshop on hCG and Related Molecules

Fragmentation of the β-Subunit of Human Chorionic Gonadotropin Produced by Choriocarcinoma*

Affinity to lectin, biological and immunological characteristics of human chorionic gonadotropins from pregnant women and trophoblastic tumour patients

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