Molecular composition of two different batches of urofollitropin: Analysis by immunofluorimetric assay, radioligand receptor assay and in vitro bioassay

1993 ◽  
Vol 16 (1) ◽  
pp. 21-27 ◽  
Author(s):  
M. Simoni ◽  
G. F. Weinbauer ◽  
E. Nieschlag
Keyword(s):  
Molecular Composition ◽  
In Vitro Bioassay ◽  
Receptor Assay ◽  
Radioligand Receptor Assay

The International Standard for Pituitary FSH: Collaborative study of the Standard and of four other purified human FSH preparations of differing molecular composition by bioassays, receptor assays and different immunoassay systems

1989 ◽  
Vol 123 (2) ◽  
pp. 275-293 ◽  
Author(s):  
P. L. Storring ◽  
Gaines Das R. E.
Keyword(s):  
Geometric Mean ◽  
In Vitro Bioassay ◽  
International Standard ◽  
Receptor Assay ◽  
Specific Activities ◽  
In Vivo Bioassay ◽  
Ranking Order ◽  
Receptor Assays

ABSTRACT The International Standard for Pituitary FSH (IS; in ampoules coded 83/575) was assayed in terms of the Second International Reference Preparation of Human Pituitary FSH and LH for Bioassay (IRP 78/549) by 27 laboratories in 13 countries using bioassays, receptor assays and immunoassays. Estimates of the FSH content of the IS by in-vivo bioassay were homogeneous both within and between laboratories and gave a combined geometric mean (with 95% fiducial limits) of 79·9 (74·6–85·4) i.u./ampoule. Estimates by different in-vitro bioassays and receptor assays were also homogeneous between assays and laboratories, and gave a combined geometric mean (with 95% fiducial limits) of 31·2 (28·8–33·9) i.u./ampoule. However, estimates by the 19 different immunoassay systems were heterogeneous and varied between 5 and 31 i.u./ampoule. The material in ampoules coded 83/575 was established by the World Health Organization as the International Standard for Pituitary FSH. It was assigned a unitage of 80 i.u./ampoule on the basis of its calibration by in-vivo bioassay, because this assay best identifies and defines the hormone. However, the introduction of the new IS will necessitate the recalibration of immunoassay kits. FSH 84/530, prepared in the same way as the IS from the same FSH preparation, did not differ significantly from the IS in any of the assay systems studied and appeared to be equally suitable as a standard. Four highly purified preparations of human FSH (FSH A–D), differing in their isoform compositions and in their in-vivo: in-vitro bioactivity ratios, were also studied. The ranking order of the specific activities of FSH A–D by in-vitro bioassays paralleled their order by receptor assays and the order of their content of FSH isoforms with isoelectric points > 4·5. (Potency estimates of FSH B and C in terms of the IS were greater by receptor assay than by in-vitro bioassay.) The overall ranking order of the specific activities of FSH A–D by immunoassays was different. Contrary to expectation, estimates in terms of the IS of specific activities by immunoassay differed more between preparations than those by in-vitro bioassay or receptor assay. Differences in specificity between immunoassay systems were demonstrated not only in the calibration of the IS in terms of the crude FSH of IRP 78/549 but also in the comparisons of the highly purified FSH in the IS and FSH A–D. The differences in the immunoreactivities and bioactivities of FSH preparations differing in their isoform compositions greatly complicate the standardization of assays for FSH. Journal of Endocrinology (1989) 123, 275–293

RADIOLIGAND RECEPTOR ASSAY OF FSH

1976 ◽  
Vol 82 (3) ◽  
pp. 673-682 ◽  
Author(s):  
P. S. Brown ◽  
R. M. Sharpe ◽  
M. Hartog ◽  
M. Shahmanesh
Keyword(s):  
Dose Response ◽  
Testicular Tissue ◽  
Rat Serum ◽  
Linear Dose ◽  
Receptor Assay ◽  
Chloramine T ◽  
Radioligand Receptor Assay

ABSTRACT The assay of FSH by a radioligand receptor assay (RLA) using homogenates of rat testicular tissue incubated for 17 h at 21°C has been assessed. Chloramine-T or lactoperoxidase were used for iodination. The assay gave linear dose-response lines between 30 and 2000 ng sheep FSH/tube, and there was usually no major interference by LH. Two batches of labelled FSH, however, gave assays in which LH showed a striking interaction with FSH. When these batches were avoided and FSH and LH were mixed in ratios that differed less than fourfold, the assay was combined successfully, in the same tubes, with an RLA for LH, using LH and FSH labelled with 131I and 125I respectively. The RLA for FSH was not suitable for assay of FSH in rat serum because of apparent non-specific interference. Assay by RLA of rat FSH, in pituitary homogenates or released during incubation in vitro, gave results which were not closely correlated with those of either conventional bioassay or radioimmunoassay.

Biological and immunological properties of the international standard for FSH 83/575: isoelectrofocusing profile and comparison with other FSH preparations

1993 ◽  
Vol 128 (3) ◽  
pp. 281-288 ◽  
Author(s):  
Manuela Simoni ◽  
Friedrich Jockenhövel ◽  
Eberhard Nieschlag
Keyword(s):  
Monoclonal Antibodies ◽  
Isoelectric Focusing ◽  
Good Correspondence ◽  
Male And Female ◽  
International Standard ◽  
Human Male ◽  
Receptor Assay ◽  
Reference Preparation ◽  
Radioligand Receptor Assay

The new international standard for FSH, IS 83/575, has been analyzed, after isoelectric focusing separation, by Sertoli cell in vitro bioassay, radioligand receptor assay and two highly specific immunometric assays. Its molecular composition was then compared with the isoelectric focusing profiles obtained from the fractionation of the reference preparation 2nd IRP 78/549 and from pools of human male and female pituitary extracts and male and female sera. The results showed that >80% of immunoreactive and bioactive FSH in the IS 83/575 has a pI value <4, while such very acidic material was represented much less in the other FSH preparations tested. All the immunoreactive material contained in the IS 83/575 was shown to be capable of receptor binding and bioactivity in vitro. A generally good correspondence between IEF profiles obtained by bioassay and by immunofluorimetric assay was evident in the case of IS 83/575, 2nd IRP 78/549 and pituitary extracts, although the profiles recorded by immunofluorimetric assay were rather smooth and more isoforms were detected by bioassay. A striking discrepancy between immunoreactive FSH and bioactive FSH was observed after isoelectric focusing fractionation of the serum pools, in which some bioactive material was not detected by immunofluorimetric assay and some of the immunoreactive FSH peaks were devoid of bioactivity, indicating that serum contains inhibitors of FSH action and that immunometric assays based on monoclonal antibodies may miss some bioactive FSH isoforms. Taken together, these results suggest that the IS 83/575 is not fully representative of pituitary and serum FSH, and its use for calibration of modern immunometric methods based on monoclonal antibodies is unlikely to resolve current problems of inaccuracy in measurements of serum FSH.

DISCREPANCY BETWEEN RADIOIMMUNOASSAY AND RADIOLIGAND-RECEPTOR ASSAY OF LUTEINIZING HORMONE RELEASED IN VITRO BY PITUITARY TISSUE FROM MALE RATS OF DIFFERENT AGES

1975 ◽  
Vol 65 (2) ◽  
pp. 265-273 ◽  
Author(s):  
R. M. SHARPE ◽  
M. SHAHMANESH ◽  
M. G. ELLWOOD ◽  
M. HARTOG ◽  
P. S. BROWN
Keyword(s):  
Luteinizing Hormone ◽  
Close Agreement ◽  
Male Rats ◽  
Male Rat ◽  
Receptor Assay ◽  
Pituitary Glands ◽  
Lh Release ◽  
Radioligand Receptor Assay ◽  
Lh Rh

SUMMARY Anterior pituitary glands from male rats aged 21, 40, 60 or 95 days were incubated in medium containing 0, 2 or 20 ng luteinizing hormone-releasing hormone (LH-RH)/ml. Incubates were assayed for LH by radioimmunoassay (RIA), by the radioligand-receptor assay (RLA) using testicular homogenates as the source of receptor and, in some instances, by the ovarian ascorbic acid depletion assay (OAAD). Irrespective of the dose of added LH-RH, glands from rats aged 40 and 60 days always showed a higher release of LH, as determined by RLA, than glands from animals aged 21 or 95 days. Measurement by RIA showed a similar pattern to RLA in the basal release of LH, but in the presence of LH-RH showed little difference in LH release by glands from rats aged 40, 60 or 95 days. The LH release caused by the higher concentration of LH-RH was always greater when measured by RLA than by RIA. Assay of comparable incubates by OAAD showed close agreement with RLA estimates in four incubations (mean index of discrimination 1·07; range 0·86–1·18) and consistent disagreement with RIA estimates (1·64; range 1·38–1·99). In contrast to the results with incubates, homogenates of pituitary glands from male rats of various ages showed close agreement of estimates by RLA, RIA and OAAD. These results suggest that RIA underestimates the LH-RH-stimulated release of LH in vitro from the male rat pituitary during some stages of sexual maturation.

Development of a radioligand receptor assay for measuring follitropin in serum: application to premature ovarian failure

1991 ◽  
Vol 37 (4) ◽  
pp. 508-514 ◽  
Author(s):  
Alan L Schneyer ◽  
Patrick M Sluss ◽  
Randall W Whitcomb ◽  
Janet E Hall ◽  
William F Crowley ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Premature Ovarian Failure ◽  
Ovarian Failure ◽  
Fsh Receptor ◽  
Serum Samples ◽  
Receptor Assay ◽  
Radioligand Receptor Assay ◽  
Fsh Bioactivity ◽  
Free Serum

Abstract We have developed a radioligand receptor assay (RRA) with sufficient sensitivity and specificity for quantifying follitropin (FSH) in unextracted serum samples. Standard curves prepared by adding pituitary FSH to either buffer or gonadotropin-free serum were parallel and statistically indistinguishable in this assay, whereas gonadotropin-free serum alone had no activity. Cross-reactivity with related pituitary hormones was negligible. Pituitary FSH was calibrated with commonly used reference preparations so that RRA results could be compared with RIA results for identical standards. The patterns in daily blood samples in six normal menstrual cycles were similar by both methods. The mean RIA:RIA ratio in both the follicular and luteal phases was between 0.6 and 0.7, and at mid-cycle decreased to 0.48, suggesting an alteration of isohormone composition at mid-cycle. In 27 women with premature ovarian failure, RRA:RIA ratios ranged from below the RRA minimum detectable dose to 4.6, suggesting that immunoreactive FSH might not be capable of binding to the FSH receptor in some patients, whereas in patients with high RRA:RIA ratios, circulating inhibitors of FSH receptor binding might be present and perhaps contributing to the observed ovarian failure. Use of this RRA in conjunction with RIA and in vitro bioassays may better define the relative contribution of FSH isohormones, autocrine or paracrine modulators of FSH bioactivity, and FSH-receptor binding competitors to the "total FSH biological signal" as detected by the gonadal FSH receptor.

Establishment of an in vitro bioassay and radio receptor assay for LH/CG in human sera using immortalized granulosa cells transfected with LH/CG receptor

Endocrine ◽  
1996 ◽  
Vol 5 (3) ◽  
pp. 275-283 ◽  
Author(s):  
Natarajagounder Selvaraj ◽  
Ada Dantes ◽  
Roma Limor ◽  
Avraham Golander ◽  
Abraham Amsterdam
Keyword(s):  
Granulosa Cells ◽  
In Vitro Bioassay ◽  
Receptor Assay ◽  
Human Sera

Biopotency in vitro and metabolic clearance rates of five pituitary preparations of follicle stimulating hormone

1993 ◽  
Vol 5 (2) ◽  
pp. 181 ◽  
Author(s):  
DJ Phillips ◽  
NL Hudson ◽  
S Lun ◽  
LA Condell ◽  
KP McNatty
Keyword(s):  
Clearance Rate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Follicle Stimulating Hormone ◽  
Metabolic Clearance ◽  
Clearance Rates ◽  
Metabolic Clearance Rate ◽  
In Vitro Bioassay ◽  
Receptor Assay ◽  
Stimulating Hormone

Five pituitary preparations of follicle stimulating hormone (FSH), namely NIDDK-oFSH-17, Bioscan oFSH, Ovagen, Folltropin-V and F.S.H.-P., were examined for biological activity in terms of their potency in an in vitro bioassay, receptor assay and heterologous radioimmunoassay and in terms of their metabolic clearance rates. In the three assays, Bioscan oFSH was the most potent (P < 0.05) (3- to 5-fold the potency of NIDDK-oFSH-17), with Ovagen being 25-50% the potency of the NIDDK standard (P < 0.05). Folltropin-V and F.S.H.-P. had the lowest potencies in all three assays. For each preparation, the ratio of activities between the assays was not consistent, suggesting that the preparations behaved differently in each assay. In 9 of 10 cases, potency estimates in the heterologous radioimmunoassay were greater than those in the in vitro bioassay or receptor assay. Polyacrylamide gel electrophoresis of the preparations showed banding consistent with the molecular weight of FSH, but also indicated that the preparations were contaminated with other proteins to varying extents. The half-lives of these preparations when injected into the bloodstream of mature female mice were 28.0, 8.6, 13.4, 11.6 and 17.4 min for NIDDK-oFSH-17, Bioscan oFSH, Ovagen, Folltropin-V and F.S.H.-P. respectively. The slopes of the decay rates were significantly different from each other (P < 0.05) except between Ovagen and Folltropin-V. The results of these studies show that a number of widely available FSH preparations have differing biopotencies. Moreover, the biopotency of a preparation in vitro is not related to its metabolic clearance rate, and not all FSH preparations behave identically in different assays. Measures of biopotency in vitro combined with those of metabolic clearance rate may provide useful information on the properties of FSH preparations used for research purposes and for superovulation of farmed livestock.

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