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.

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

Immunoreactive β-core-like material in normal postmenopausal urine: human chorionic gonadotrophin or LH origin? Evidence for the existence of LH core

1992 ◽  
Vol 133 (3) ◽  
pp. 459-466 ◽  
Author(s):  
R. K. Iles ◽  
C. L. Lee ◽  
I. Howes ◽  
S. Davies ◽  
R. Edwards ◽  
...  
Keyword(s):  
Postmenopausal Women ◽  
Molecular Size ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Core Material ◽  
Β Subunit ◽  
Α Subunit ◽  
Serum Lh ◽  
Exclusion Chromatography ◽  
Β Hcg

ABSTRACT Material with the immunochemical properties of the β-core of human chorionic gonadotrophin (hCG) can be found in the urine of normal postmenopausal women. However, we have been unable to detect intact hCG (using an assay which is specific for the α–β heterodimer of intact hCG) in serum of such subjects. The levels of serum LH and urinary β-core were compared in matched samples from 28 women (serum LH: median 27 U/l, range 4-70 U/l, urinary β-core: median 0·27 μg/l, range < 0·05–0·645 μg/l). Urine (4 litres) from three postmenopausal women was concentrated, dialysed and subjected to gel exclusion chromatography on Sephadex G-100. Fractions were analysed by specific assays for LH, intact hCG, total β-hCG (free β-subunit and intact hCG), free α-subunit and β-core. Material eluting at the expected position of the β-core fragment of hCG was detected in all three samples by the β-core, β-hCG and LH assays, despite the fact that the LH antibody does not recognize the authentic β-core of pregnancy. Electrophoresis and Western blotting of the concentrated urines revealed that material of the same molecular size as β-core was recognized by the antibody to LH but not by a monoclonal antibody raised to free β-hCG which also recognizes the β-core molecule of hCG. We conclude that the predominant core-like material identified in postmenopausal urine is probably derived from the β-subunit of LH. Journal of Endocrinology (1992) 133, 459–466

Lysine residues 2 and 104 of the human chorionic gonadotrophin β subunit influence receptor binding

1993 ◽  
Vol 10 (3) ◽  
pp. 337-343 ◽  
Author(s):  
H Xia ◽  
J Huang ◽  
T-M Chen ◽  
D Puett
Keyword(s):  
Amino Acid ◽  
Receptor Binding ◽  
Basic Amino Acid ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Amino Acid Residues ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Lysine Residues

ABSTRACT Human chorionic gonadotrophin (hCG), like other members of the glycoprotein hormone family, contains a common α subunit and a hormone-specific β subunit. The latter is a 145 amino acid residue polypeptide with six sites of glycosylation. Positions 2 and 104 are occupied by basic amino acid residues in the 12 known amino acid sequences of mammalian β subunits from CG and LH, a related gonadotrophin that acts through the same receptor. Lysine residues are found in both these positions in hCG-β. Using site-directed mutagenesis, each of these two lysines in hCG-β was replaced with glutamic acid. The mutant and wild-type cDNAs were subcloned into a eukaryotic expression vector, which was then transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for the bovine α subunit. Holoprotein formation occurred with each of the two heterologous gonadotrophin mutants, i.e. the bovine α subunit bound to hCG-β (Glu2) and to hCG-β (Glu104), as well as with the control, i.e. the bovine α subunit bound to the hCG-β wild-type subunit. In two in-vitro assays, one a competitive binding assay with 125I-labelled hCG as bound ligand and the other based on stimulation of progesterone production in a transformed murine Leydig cell line, MA-10, both the heterodimers containing a mutant β subunit exhibited bioactivity, but their potencies were lower than that of the bovine α subunit bound to the hCG-β wild-type subunit. These results suggest that the basic amino acid residues at positions 2 and 104 in hCG-β participate, either directly or indirectly, in receptor binding.

Monoclonal antibodies against the free subunits of human chorionic gonadotrophin

1990 ◽  
Vol 125 (2) ◽  
pp. 301-309 ◽  
Author(s):  
P. Berger ◽  
R. Klieber ◽  
W. Panmoung ◽  
S. Madersbacher ◽  
H. Wolf ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cross Reactivity ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Antigenic Determinants ◽  
Α Subunit ◽  
Α Chain ◽  
Fluid Levels ◽  
Α Subunits

ABSTRACT Discordant results on body fluid levels of human chorionic gonadotrophin (hCG) free α- and β-subunits under physiological and pathophysiological conditions, prompted us to raise a total of 260 monoclonal antibodies (MCA) against free hCG-α, free hCG-β, holo-hCG, human follicle-stimulating hormone and bovine luteinizing hormone; 153 MCA recognizing the human α-subunit and 28 reacting with hCG-β were extensively analysed for their intra- and interspecies cross-reactivity with homologous hormones, and for the compatibility of epitopes recognized by them. The immunological topography of free hCG-α and free hCG-β was resolved by these MCA, and epitope maps were designed. Six antigenic determinants on the free α-chain (α1–α6), clustered in three spatially distinct domains, and seven epitopes on the surface of free hCG-β (β1–β7), could be distinguished. Strikingly, three α-chain epitopes (α4, α5 and α6) were shared between various species, which is in contradiction to the concept of immunological species-specificity of α-subunits. Three determinants were found to be present only on the free subunits but not on holo-hCG (α6, β6 and β7), and only two determinants (β1 and β7) were hormone-specific for hCG. Based on this information, an immunoenzymometric assay for the free α-subunit of human glycoprotein hormones was established, with a sensitivity of 1·3 pg/well and a cross-reactivity with holo-hCG of less than 0·005% Thus this assay provides the basis for detecting free α-subunits in the presence of extremely high levels of holo-hormones, which may assist in elucidating the role of free α-subunits in man. Journal of Endocrinology (1990) 125, 301–309

Consequences of antisense human chorionic gonadotrophin-α subunit cDNA expression in human choriocarcinoma JAR cells

1995 ◽  
Vol 14 (3) ◽  
pp. 337-347 ◽  
Author(s):  
H Cao ◽  
Z M Lei ◽  
Ch V Rao
Keyword(s):  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Mrna Levels ◽  
Β Subunit ◽  
Α Subunit ◽  
Transfected Cells ◽  
Subunit Mrna ◽  
Empty Vector ◽  
Cdna Expression ◽  
Jar Cells

ABSTRACT The biosynthesis of human chorionic gonadotrophin (hCG) is a hallmark endocrine function of human choriocarcinoma cells. The present study investigated the consequences of greatly diminishing this synthesis in JAR cells by stably transfecting them with pRSV-antisense hCG-α cDNA expression vector. The vector directs the synthesis of antisense hCG-α subunit mRNA which would then bind to sense hCG-α subunit mRNA, thus blocking its translation and consequently dimer hCG protein synthesis. The transfection with pRSV-antisense hCG-α cDNA resulted in a dramatic decrease in hCG secretion as compared with untransfected parental cells or those transfected with an empty vector used for the selection of clones. The decreased secretion was due to a decreased synthesis which in turn was due to a fall in steady-state hCG-α and -β subunit mRNA levels. The decrease of hCG-β subunit transcripts was unexpected and it was not due to contamination of antisense hCG-α cDNA construct with hCG-β sequence. The transcription of hCG-α and -β subunit genes was not altered in transfected cells suggesting that increased degradation was responsible for decreased steadystate hCG subunit mRNA levels. Despite the decreased hCG levels, the transfected cells maintained normal hCG receptor levels, responded to epidermal growth factor stimulation of hCG synthesis and secretion and grew at the same rate as the control parental cells and those transfected with an empty vector.

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.

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