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.

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.

RELATIONSHIPS BETWEEN PHYSICO-CHEMICAL, IMMUNOLOGICAL AND BIOLOGICAL PROPERTIES OF HUMAN CHORIONIC GONADOTROPHIN

1971 ◽  
Vol 67 (3) ◽  
pp. 434-444 ◽  
Author(s):  
D. L. Matthies ◽  
E. Diczfalusy
Keyword(s):  
Biological Activity ◽  
Gel Filtration ◽  
First Trimester ◽  
Biological Properties ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Separate Component ◽  
Immunological Activity ◽  
Physico Chemical ◽  
Pregnancy Urine

ABSTRACT Gel filtration of pregnancy urine on Sephadex G-100 separates two molecular species related to human chorionic gonadotrophin (HCG). One is a slightly retarded fraction which possesses biological activity but little or no immunological activity. The other is a highly retarded fraction which possesses immunological activity but no detectable biological activity. The highly retarded material could not be detected in pregnancy serum nor in extracts of first trimester placentas. A nonpregnant woman injected intravenously with purified HCG which displayed only slight retardation on the column produced urine which contained the highly retarded material. Experiments are described, the results of which suggest that the highly retarded material is a separate component of urine, probably generated by the kidney.

scholarly journals Cross-reaction of human chorionic gonadotrophin in Immulite 2000 luteinizing hormone assay

2002 ◽  
Vol 39 (3) ◽  
pp. 318-319
Author(s):  
S Vivekanandan ◽  
Charles E Andrew
Keyword(s):  
Luteinizing Hormone ◽  
Pregnant Women ◽  
Cross Reactivity ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Cross Reaction ◽  
Positive Bias ◽  
The Cross ◽  
Spiked Serum ◽  
Hormone Assay

Background and method The cross-reactivity of human chorionic gonadotrophin (hCG) in the new DPC Immulite 2000 luteinizing hormone immunoassay was studied using sera from healthy pregnant women and pooled serum spiked with hCG standard IRP 75/537. Results and Conclusion Significant positive bias was seen in sera from pregnant women and in IRP 75/537 spiked serum.

SUBUNITS OF HUMAN CHORIONIC GONADOTROPHIN: AN IMMUNOCHEMICAL STUDY

1973 ◽  
Vol 73 (1) ◽  
pp. 133-145 ◽  
Author(s):  
S. Donini ◽  
V. Olivieri ◽  
G. Ricci ◽  
P. Donini
Keyword(s):  
Gel Filtration ◽  
Cross Reactivity ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Physical Characterization ◽  
Human Pituitary ◽  
Immunochemical Study ◽  
Urea Treatment ◽  
Α And Β Subunits

ABSTRACT The α and β subunits of HCG were prepared by urea-treatment of the hormone and isolated by DEAE-Sephadex A-25 chromatography. Further purification of the subunits was achieved by gel filtration on Sephadex G-100. Partial chemico-physical characterization confirmed the non-identity of the HCG subunits, as previously reported by several investigators. A radioimmunoassay (RIA) system based on 125I-HCG-β + anti-HCG-β serum was used to study the antigenic similarities between HCG, HCG-α and -β, human pituitary LH, urinary LH and FSH. The highest cross-reactivity was found for HCG, followed by HCG-α, LH and FSH. The specificity of the above mentioned RIA system was increased by absorbing the anti-HCG-β serum with HCG-α. Because of the ability of this absorbed antiserum to discriminate between HCG and LH, a study was carried out to detect specifically HCG in urine in spite of the presence of physiological amounts of LH.

COMPARISON OF BIOASSAY AND IMMUNOASSAY OF HUMAN CHORIONIC GONADOTROPHIN IN URINE

1965 ◽  
Vol 50 (3) ◽  
pp. 335-344 ◽  
Author(s):  
Rudi Borth ◽  
Michel Ferin ◽  
Annette Menzi
Keyword(s):  
Biological Activity ◽  
Total Variation ◽  
Regression Line ◽  
Haemagglutination Inhibition ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Factorial Experiment ◽  
Serological Result ◽  
Pregnancy Urine ◽  
Acceptable Agreement

ABSTRACT In 39 samples of pregnancy urine, the concentration of human chorionic gonadotrophin (HCG) was estimated biologically by the ovarian hyperaemia reaction in rats, and serologically by the passive haemagglutinationinhibition technique. The results of the bioassays varied from 3 to 150 IU/ml, those of the immunoassays from 5 to 640 IU-eq./ml, and the correlation between the two (calculated for their logarithms) accounted for only 17 per cent of the total variation (r2 = 0.169, P ≈0.01). If the biological activity were estimated from a serological result and the appropriate regression line, the fiducial interval for P = 0.05 would extend from 17 to 610 per cent of the estimate. In a factorial experiment using three anti-HCG sera, three standard and three sensitizing preparations of HCG, the sensitivity of the serological system (expressed as the endpoint concentration in IU of HCG) varied considerably between the 27 combinations of the 3 factors, but there was no interaction between the latter. From these data and those of other authors, it is concluded that immunoassays based on haemagglutination inhibition cannot replace bioassays in the estimation of HCG, as distinct from its hypothetical metabolites or other related antigens, unless specificity has been demonstrated. The well-documented reliability of serological pregnancy tests is, of course, not in dispute. Attention is drawn to the fact that »statistically significant« correlation does not guarantee analytically acceptable agreement between two methods of assay.

ANTIGONADOTROPHIC PROFILE OF ANTISERA AGAINST HUMAN GONADOTROPHIN PREPARATIONS

1971 ◽  
Vol 67 (2) ◽  
pp. 262-276 ◽  
Author(s):  
P. Petrusz ◽  
C. Robyn ◽  
E. Diczfalusy
Keyword(s):  
Biological Activity ◽  
Luteinizing Hormone ◽  
Total Dose ◽  
Follicle Stimulating Hormone ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Immunological Activity ◽  
Human Menopausal Gonadotrophin ◽  
Stimulating Hormone

ABSTRACT Forty-two antisera were prepared in rabbits against human chorionic gonadotrophin (HCG), human hypophysial gonadotrophin (HHG), human urinary luteinizing hormone (LH) and human menopausal gonadotrophin (HMG) preparations. The gonadotrophic profiles of the antigens were previously characterized by bioassay, immunoassay and bioimmunoassay methods. The 25 most potent antisera were tested in statistically valid bioassays for their HCG and follicle stimulating hormone (FSH) neutralizing activities as well as for their neutralizing potencies against the FSH-like activity present in HCG preparations. The anti-HCG/anti-FSH ratios of the anti-HCG sera tested varied between 6.2 and > 254, while those of the anti-HHG, anti-LH and anti-HMG sera were close to 2. It was found that the total dose of immunological activity (anti-HCG neutralizing and anti-FSH neutralizing potency) rather than that of the biological activity administered to the rabbits was decisive for obtaining antisera with high anti-HCG and anti-FSH titers. Immunization with a highly purified HCG preparation (> 17 000 IU/mg) resulted in antisera exhibiting lower anti-HCG/anti-FSH ratios than did immunization with partially purified preparations. A highly purified urinary LH preparation which did not contain any detectable FSH activity gave rise to antisera exhibiting anti-HCG/anti-FSH ratios of approximately 2.0. These highly purified HCG and LH preparations were shown previously to possess high anti-FSH neutralizing potencies (Petrusz et al. 1971b). Booster injections did not change significantly the quality or the titer of the antigonadotrophic sera studied. The HCG neutralizing potency of anti-HCG sera was approximately 3 times higher when assayed against a highly purified HCG preparation (> 17 000 IU/mg) as compared to potency estimates obtained against the laboratory standard of HCG (about 2000 IU/mg). It is suggested that consideration should be given to the establishment of standard preparations of antigonadotrophic sera. It is concluded that bioimmunoassays are more suitably than conventional bioassay methods for the assessment of the antigenic purity of human gonadotrophin preparations.

STUDIES ON A HUMAN CHORIONIC GONADOTROPHIN-LIKE MATERIAL PRESENT IN NON-PREGNANT SUBJECTS

1978 ◽  
Vol 89 (3) ◽  
pp. 492-505 ◽  
Author(s):  
D. M. Robertson ◽  
H. Suginami ◽  
H. Hernandez Montes ◽  
C. P. Puri ◽  
S. K. Choi ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Specific Sequence ◽  
Gonadotrophin Secretion ◽  
Carboxy Terminal Region ◽  
Assay Methods ◽  
Low Levels ◽  
Carboxy Terminal

ABSTRACT The presence of an hCG-like material in urinary and pituitary extracts and plasma obtained from non-pregnant subjects was investigated. Two assay methods were used to detect this material following fractionation of pituitary and urinary extracts by gel filtration (Ultrogel AcA 54) and/or isoelectrofocusing: a) a radioimmunoassay employing an antiserum raised against a specific sequence of the carboxy terminal region (residues 115– 145) of the β-subunit of hCG, and b) an in vitro bioassay method which measures both hLH and hCG activities. The fractionation procedures employed provide a satisfactory separation of highly purified hCG and hLH preparations. In the pituitary and urinary extracts hCGβ-peptide-like immunoactive (PIA) material was found consistently, which co-eluted with iodinated hCG following gel filtration and possessed pI values similar to those of hCG when subjected to isoelectrofocusing. The PIA material also exhibited in vitro biological activity similar to that shown by hLH and hCG. Detectable levels of immunoactive material were also found in plasma; however, the plasma levels of this PIA material were not influenced by classical endocrine measures such as the stimulation or inhibition of gonadotrophin secretion. The low levels of this material in plasma precluded its further characterization by gel filtration or electrofocusing. Whereas the present data and those reported by other investigators seem to suggest the presence of some hCG-like material in urinary and pituitary extracts and possibly in plasma of non-pregnant subjects, it is emphasized that the available evidence is not sufficiently conclusive to exclude other interpretations as to the nature of this material.

A COMPARISON BETWEEN CHORIONIC GONADOTROPHINS EXTRACTED FROM HUMAN, RHESUS MONKEY AND MARMOSET PLACENTAE

1972 ◽  
Vol 55 (2) ◽  
pp. 363-368 ◽  
Author(s):  
BRUCE HOBSON ◽  
LEIF WIDE
Keyword(s):  
Biological Activity ◽  
Rhesus Monkey ◽  
Human Placenta ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Antigenic Determinants

SUMMARY Evidence is provided to show that chorionic gonadotrophins extracted from the human, rhesus monkey and marmoset placentae have antigenic determinants in common. Similar slopes were obtained for these gonadotrophins in a radioimmunoassay for human chorionic gonadotrophin (HCG). The biological activity of the monkey gonadotrophins was neutralized by anti-HCG serum. When the gonadotrophic activity of the monkey placental extracts was assayed biologically and immunologically, using HCG as a standard, similar results were obtained. Higher values were obtained by the immunoassay than by the bioassay when extracts of human placenta were assayed using the same HCG standard.

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