Monoclonal antibodies to the β-subunit of human chorionic gonadotrophin

1988 ◽  
Vol 106 (6) ◽  
pp. 1727-1729
Author(s):  
R. Fidler ◽  
A. V. Sokolov ◽  
M. Sh. Verbitskii ◽  
I. P. Papazov
Keyword(s):  
Monoclonal Antibodies ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Β Subunit

The production of monoclonal antibodies to human chorionic gonadotrophin and its subunits

1983 ◽  
Vol 98 (3) ◽  
pp. 323-330 ◽  
Author(s):  
M. C. Stuart ◽  
P. A. Underwood ◽  
D. F. Harman ◽  
K. L. Payne ◽  
D. A. Rathjen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Free Form ◽  
Binding Affinities ◽  
Β Subunit ◽  
Α Subunit ◽  
Specific Antisera ◽  
Hybridoma Technology ◽  
Circulating Levels

The difficulties encountered in producing highly specific antisera to human chorionic gonadotrophin (hCG) were overcome by the use of hybridoma technology. A panel of monoclonal antibodies directed toward hCG and its subunits was produced. Of the four antibodies which were fully characterized, one recognized the intact hCG molecule only, a second recognized only the free β-subunit, a third recognized only the free α-subunit and the fourth bound to the β-subunit of hCG both when it was in the free form and when it was associated with the α-subunit forming the intact hCG molecule. There was no significant cross-reaction of any of these antibodies with the pituitary glycoprotein hormones. The four antibodies had high binding affinities which should permit their use in immunoassays for measurement of circulating levels of hCG and its subunits.

Characteristics of Monoclonal Antibodies Against Human Chorionic Gonadotrophin

1982 ◽  
pp. 837-842 ◽  
Author(s):  
A.M.G. BOSCH ◽  
W. STEVENS ◽  
A. SCHUURS ◽  
O. SCHÖNHERR ◽  
H. ROELOFS
Keyword(s):  
Monoclonal Antibodies ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin

A single amino acid residue replacement in the β subunit of human chorionic gonadotrophin results in the loss of biological activity

1992 ◽  
Vol 8 (1) ◽  
pp. 87-89 ◽  
Author(s):  
F. Chen ◽  
D. Puett
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Β Subunit ◽  
Single Amino Acid Residue

ABSTRACT The heterodimer, human chorionic gonadotrophin (hCG), contains an a subunit that is common to the glycoprotein hormones and a hormone-specific β subunit. A comparison of all known β amino acid sequences shows that an aspartic acid at position 99 (with the numbering scheme for hCG-β) is one of the seven non-Cys invariant residues. Using site-directed mutagenesis we have replaced hCG-β Asp99 with Arg. Chinese hamster ovary cells, containing a stably integrated gene for bovine a subunit, were transiently transfected with plasmids containing wild-type and mutant hCG-β cDNAs. The Arg99 β mutant associated with the a subunit, but the resulting heterodimer failed to enhance intracellular cyclic AMP production in a gonadotrophin-responsive transformed murine Leydig cell line. Thus, a single amino acid residue replacement in this glycosylated heterodimer containing 237 amino acid residues is sufficient to abolish activity.

scholarly journals Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin β-subunit

2012 ◽  
Vol 18 (8) ◽  
pp. 379-390 ◽  
Author(s):  
Liina Nagirnaja ◽  
Česlovas Venclovas ◽  
Kristiina Rull ◽  
Kim C. Jonas ◽  
Hellevi Peltoketo ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Missense Mutations ◽  
Β Subunit

Total amounts of circulating human chorionic gonadotrophin α and β subunits can be assessed throughout human pregnancy using immunoradiometric assays calibrated with the unaltered and thermally dissociated heterodimer

1994 ◽  
Vol 140 (3) ◽  
pp. 513-520 ◽  
Author(s):  
A-M Nagy ◽  
D Glinoer ◽  
G Picelli ◽  
J Delogne-Desnoeck ◽  
B Fleury ◽  
...  
Keyword(s):  
Early Pregnancy ◽  
Thermal Dissociation ◽  
Heat Exposure ◽  
Free Fraction ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Β Subunit ◽  
Human Pregnancy ◽  
Molar Basis ◽  
Α And Β Subunits

Abstract The aim of the present study was to determine the variations in the balance between total (free plus combined) circulating α and β subunits of human chorionic gonadotrophin (hCG) throughout human pregnancy. The equivalence between the International Units (IU) of hCG (IRP 75/537) and those assigned to the α (IRP 75/569) and β (IRP 75/551) free subunits was experimentally determined by using intact and thermally dissociated hCG. Heat exposure (2 min at 100 °C) of hCG preparations resulted in a complete dissociation of hCG into free, soluble and intact α and β subunits. The hCG and α and β subunit contents of unaltered and heated hCG preparations were assessed by specific immunoradiometric assays. The amount of immunoreactive subunits dissociated by heat from hCG could then be evaluated on a molar basis. Circulating hCG and its free α and β subunits were immunoassayed in 836 blood samples collected from healthy pregnant women at different gestational ages. After conversion of hCG and its subunits into a common IU system, the gestational profiles of the total amounts (free plus combined) of α- and βhCG subunits increased together and peaked at 9–10 weeks of gestation. Thereafter, total α and β subunits decreased and subsequently remained stable until term. The decline in total αhCG subunit was less marked than that of total βhCG subunit. The α- to βhCG ratio was equimolar until 10 weeks of gestation when it increased almost fourfold until term (P<0·0001). Finally, the free fraction of the total circulating αhCG subunit represented 5–7% in early pregnancy but reached 60–70% in the last trimester (P<0·0001). In contrast, the free fraction of the total βhCG subunit decreased slightly from 4–5% of total β subunit in early pregnancy to 2–3% at term (P<0·0001). The present study suggests that thermal dissociation of hCG is a useful method with which to calibrate immunoassays on a molar basis in order to assess circulating levels of the heterodimer and its subunits. Journal of Endocrinology (1994) 140, 513–520

The molecular basis for epitopes on the free β-subunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide and the hCGβ-core fragment

1994 ◽  
Vol 141 (1) ◽  
pp. 153-162 ◽  
Author(s):  
S Dirnhofer ◽  
S Madersbacher ◽  
J-M Bidart ◽  
P B W Ten Kortenaar ◽  
G Spöttl ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Basis ◽  
Tertiary Structure ◽  
Solid Phase ◽  
Critical Role ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Antigenic Determinants ◽  
Β Subunit ◽  
Carboxyl Terminal

Abstract The molecular basis for antigenic determinants on the free β-subunit of human chorionic gonadotrophin (hCGβ), its carboxyl-terminal peptide (hCGβCTP) and the hCGβcore fragment (hCGβcf) was elucidated by means of monoclonal antibodies (MCAs). The objective of the present study was to resolve the antigenic topography of these three molecules in terms of epitope identification at different levels of structural organization as well as analysis of their spatial arrangement. An hCGβcf preparation, a synthetic peptide corresponding to the hCGβCTP (β109–145), overlapping synthetic peptides spanning the entire amino acid sequence of hCGβ, and a reduced and alkylated hCGβ preparation were assayed in a solid-phase one-site enzyme-linked immunoassay and in a solublephase direct-binding radioimmunoassay (RIA) or competitive RIA. The antigenic topography was mapped by incorporating the MCAs into two-site binding assays. On the surface of free hCGβ, nine different epitopes (β1–β9), arranged in three spatially distinct domains, could be distinguished. Epitopes β1–β7 were located in a single large domain on both hCGβ and the hCGβcf whereas hCGβCTP contained two topographically distant determinants, designated β8 and β9 respectively. All but the two epitopes located on hCGβCTP (β8 and β9) were destroyed by reducing and alkylating hCGβ, suggesting that most antigenic determinants are predominantly non-contiguous and require an intact tertiary structure whereas the molecular structure of hCGβCTP is linear. At a molecular level, amino acid residues spanning hCGβ 45–52, hCGβ 137–144 and hCGβ 113–116 contributed to the formation of epitopes β5, β8 and β9 respectively. We have also shown that the hCGβcf represents the immunodominant part of the free β-subunit of hCG, containing seven mainly conformationally determined epitopes, one of which has a share of the sequence β45–52. The hCGβCTP does not play a critical role in the immunologically important tertiary structure of hCGβ and was itself found to be a predominantly continuous sequence also within the native hormone, expressing two spatially distant antigenic determinants located within residues β113–116 and β137–144 respectively. Journal of Endocrinology (1994) 141, 153–162

SUBUNITS OF HUMAN CHORIONIC GONADOTROPHIN: IMMUNOLOGICAL AND BIOLOGICAL STUDIES

1975 ◽  
Vol 79 (4) ◽  
pp. 749-766 ◽  
Author(s):  
S. Donini ◽  
I. D'Alessio ◽  
P. Donini
Keyword(s):  
Biological Activity ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Biologically Active ◽  
Β Subunit ◽  
Reactive Material ◽  
Biological Studies ◽  
The Cross

ABSTRACT The α and β subunits of human chorionic gonadotrophin (hCG) were prepared by incubation in 8 m urea, pH 4.5. The separation of the two subunits was obtained by DEAE-Sephadex A-25 chromatography and purification was carried out by gel filtration on Sephadex G-100. The β subunit obtained was biologically active and was therefore further purified by affinity chromatography using as immuno-adsorbent the α antibodies coupled to Sepharose 4B. The β subunit so purified showed a biological activity less than 1 IU/mg. The immunological and biological properties of the hCG subunits have been studied. It was found that the anti hCG β serum can discriminate between hCG and hLH and that in the 125I-hCG + anti-β serum radioimmunoassay, the cross-reactivity of pituitary hLH was lower than that of urinary hLH. Moreover, it was observed that the less purified was the urinary LH preparation, the higher was the cross-reactivity. Therefore we considered the hypothesis that during the purification of human menopausal gonadotrophin (hMG) some LH subunits or smaller immunoreactive fragments could have been discarded with the waste fractions. In order to test the validity of this hypophysis, all the protein fractions obtained during the purification of the hMG were gel-filtered on Sephadex G-100. The immunoreactivity of the effluents from the gel filtration was tested by hCG, hCG-β, hCG-α and hLH radioimmunoassays. While the α reactive material was found in some fractions as a peak having the same Ve/Vo value as hCG-α, the β reactive material present in the crude hMG fractions was not observed in other fractions. The cross-reactivity with the anti β serum was very low and was found in the LH region of the gel chromatogram. Furthermore, the neutralization of the biological activity of hCG and of urinary and pituitary LH by the anti hCG β serum was studied by incubating a fixed amount of the three hormones with increasing volumes of antiserum and measuring the LH activity after incubation by the OAAD test. It was observed that the anti hCG β serum inhibits hCG more than urinary or pituitary LH.

A novel form of ectopic human chorionic gonadotrophin β-subunit in the serum of a woman with epidermoid cancer

1985 ◽  
Vol 107 (3) ◽  
pp. 403-408 ◽  
Author(s):  
S. B. Nagelberg ◽  
L. A. Cole ◽  
S. W. Rosen
Keyword(s):  
Molecular Weight ◽  
Unknown Origin ◽  
Apparent Molecular Weight ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Size Difference ◽  
Gel Chromatography ◽  
Β Subunit ◽  
Peanut Agglutinin ◽  
Pregnancy Urine

ABSTRACT A novel form of free human chorionic gonadotrophin β-subunit (hCGβ) was found in serum from ElBre, a woman with epidermoid carcinoma of unknown origin. ElBre hCGβ was larger than standard (pregnancy urine) hCGβ when analysed by gel chromatography (apparent molecular weight 54 000 vs 44000). This size difference appeared to be due to a larger carboxyterminal extension (CTE) of ElBre hCGβ since thermolysin cleavage of the CTE from standard hCGβ and Elbre hCGβ yielded core products of the same size. Oligosaccharides, O-linked to serine or threonine, were present in ElBre hCGβ, presumably on its CTE as judged by the complete binding of desialylated ElBre hCGβ to immobilized peanut agglutinin (this lectin is specific for terminal galactose linked β1 → 3 to N-acetylgalactosamine, a disaccharide exposed after desialylation of the O-linked oligosaccharides of standard hCGβ). ElBre hCGβ, however, was incompletely recognized by antisera specific for the CTE of standard hCGβ, especially the carbohydrate-sensitive antiserum R141. The O-linked oligosaccharides of standard hCGβ are heterogeneous in size; 13% are of the largest (hexasaccharide) form. In contrast, over 50% of the O-linked oligosaccharides in hCGβ from the JAr choriocarcinoma cell line are hexasaccharides. Like desialylated ElBre hCGβ, desialylated JAr hCGβ bound completely to peanut agglutinin, but was incompletely recognized by antisera to the hCGβ-CTE. Furthermore, JAr hCGβ was intermediate in size between standard hCGβ and ElBre hCGβ when analysed by gel chromatography (apparent molecular weight 49 000). Thus, we propose that ElBre hCGβ had an even higher proportion of large O-linked oligosaccharides than had JAr hCGβ. Moreover, the N-linked oligosaccharides of ElBre hCGβ differed from standard hCGβ; only 55% of ElBre hCGβ bound to Concanavalin A versus 89% of standard hCGβ. These data further support the concepts of aberrant glycosylation by neoplastic tissues and carbohydrate heterogeneity of hCGβ produced by various tissues. J. Endocr. (1985) 107, 403–408

NON-IDENTICAL REACTION OF UNDISSOCIATED HCG AND HCG-β SUBUNIT WITH ANTI-HCGβ SERUM

1975 ◽  
Vol 80 (2) ◽  
pp. 374-379 ◽  
Author(s):  
J. Arends
Keyword(s):  
Gel Filtration ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Β Subunit ◽  
Suitable Standard

ABSTRACT Based on gel-filtration experiments, estimates of the content are given of undissociated human chorionic gonadotrophin (HCG) and HCG-β subunit in HCG preparations. Radioimmunoassay of three HCG preparation using an antiserum against HCG-β subunit showed that the slope of displacement curves was dependent on the ratio of HCG-β subunit to undissociated HCG, the slope being steeper (negative) with increasing ratio. The implication of this observation on the choice of a suitable standard for HCG-β radioimmunoassay is discussed.

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