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.

Testicular receptors of human chorionic gonadotrophin in adult men. Binding and degradation of the hormone

1982 ◽  
Vol 101 (2) ◽  
pp. 293-300 ◽  
Author(s):  
Geneviève Grizard ◽  
Daniel Boucher ◽  
Jean Hermabessière ◽  
Jean Grizard
Keyword(s):  
Testicular Tissue ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Saturation Curve ◽  
Temperature Dependent ◽  
Adult Men ◽  
Number Of Binding Sites ◽  
Chloramine T ◽  
Substrate Concentrations

Abstract. Binding and degradation of human chorionic gonadotrophin (hCG) to testicular tissue obtained by biopsy from 9 men with gonadal disorders were investigated. Vacant hCG receptors were assayed in partially purified testicular homogenates using [125I]hCG (radio-iodinated with chloramine T). Degradation of [125I]hCG during exposure to human testicular preparations was measured in terms of the ability of supernatants to specifically bind to rat testicular receptors. Binding of [125I]hCG was time and temperature dependent. At 37°C, a maximum was reached at 8 h. It was also found to be a saturable process with respect to homogenate and hormone concentrations. Association constants and number of binding sites determined in 9 men, using Scatchard plot and saturation curve analysis ranged, respectively, from 0.2 to 1.8 × 1010m−1 and from 92 to 3427 fmol/g testis or 7 to 380 fmol/mg protein. Degradation of [125I]hCG increased with temperature and time of exposure to human testicular homogenate. It increased also with increasing human testicular homogenate concentration and substrate concentrations. For a similar concentration of [125I]hCG, per cent of degraded hormone ranged from 32 to 57, according to the subjects. These results show that human testicular homogenates are capable of binding and degrading hCG in vitro. Biological and physiological implications of degradation for hormone binding are discussed.

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.

scholarly journals In vitro validation of the tear matrix metalloproteinase 9 in-situ immunoassay

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Seung Pil Bang ◽  
Myeong Jin Son ◽  
Harim Kim ◽  
You Hyun Lee ◽  
Jong Hwa Jun
Keyword(s):  
Dose Response ◽  
Response Pattern ◽  
Sample Volume ◽  
Linear Dose ◽  
Almost All ◽  
Metalloproteinase 9 ◽  
Positive Results ◽  
Solvent Volume

Abstract We aimed to validate a tear MMP-9 in-situ immunoassay (InflammaDry) and to identify factors that could affect results or interpretation. Three factors were examined: sample concentration, volume, and time. Recombinant human (rh) MMP-9 (10 or 20 μl; 0, 12.5, 25, 50, 100, 200, 500, and 1,000 ng/ml) was applied to the kit and the detection limit and assay reproducibility were examined. At a rhMMP-9 volume of 10 μl (≥ 50 ng/ml), all positive results were identified by densitometry at 10 and 20 min; however, after 20 min, more than half of the nine ophthalmologists interpreted a positive result. At a rhMMP-9 volume of 20 μl (≥ 25 ng/ml), ophthalmologists and densitometry identified almost all test lines at 10 and 20 min. At 10 μl, densitometry showed a linear dose–response pattern. At 20 μl, densitometry showed a linear dose–response pattern at concentrations up to 500 ng/ml; however, full saturation was achieved at concentrations ≥ 500 ng/ml. When the same amount of rhMMP-9 was applied, the density result increased significantly upon doubling of the solvent volume (i.e., by adding the same volume of PBS to a sample). InflammaDry showed a high inter- and intra-assay coefficient of variation at 10 min (28.4% and 24.7%, respectively). The results of the MMP-9 in-situ immunoassay varied significantly depending on sample volume. Therefore, when interpreting the results, careful attention must be paid to tear volume.

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