Varying dose–response characteristics of different immunoassays and an in-vitro bioassay for FSH are responsible for changing ratios of biologically active to immunologically active FSH

1990 ◽  
Vol 127 (3) ◽  
pp. 523-532 ◽  
Author(s):  
F. Jockenhövel ◽  
S. A. Khan ◽  
E. Nieschlag
Keyword(s):  
Sertoli Cell ◽  
Dose Response ◽  
Serum Levels ◽  
Biologically Active ◽  
Testicular Function ◽  
In Vitro Bioassay ◽  
Great Caution ◽  
Response Characteristics ◽  
Infertile Men

ABSTRACT Serum FSH levels in fertile and infertile men were determined by applying the Sertoli cell in-vitro bioassay and six different immunoassays. Bioassay and immunoassay estimates were significantly correlated (r ranging from 0·78 to 0·86; P<0·01). On average, all immunoassays measured lower FSH concentrations in samples with low FSH levels and higher FSH concentrations in those with high FSH levels compared with the bioassay. Ratios of bioactivity to immunoreactivity (B/I) were highest in fertile men and lowest in men with severe disturbances of testicular function. Depending on which immunoassay was used these differences were either significant or only marginal. Dose–response characteristics for WHO FSH standard preparation 78/549, used in the bioassay as well as in the immunoassays, were different between immunoassays and the bioassay, suggesting that decreasing B/I ratios with increasing FSH serum levels were method-related and reflected different slopes of the dose–response characteristics of the assays, rather than being true changes in the molecular composition of FSH. The present investigation underlines the necessity of choosing the immunoassay used for comparison with the bioassay carefully and of validating the system in regard to parallelism between dose–response characteristics. B/I ratios must be interpreted with great caution and previous studies which report changing B/I ratios in various endocrine situations may have to be reevaluated. Journal of Endocrinology (1990) 127, 523–532

scholarly journals Disorders of Puberty: Endocrinology of the Pre-Pubertal Testis

2020 ◽  
Vol 9 (3) ◽  
pp. 780 ◽  
Author(s):  
Sandro La Vignera ◽  
Rossella Cannarella ◽  
Rosita A. Condorelli ◽  
Aldo E. Calogero
Keyword(s):  
Male Infertility ◽  
Sertoli Cell ◽  
Sperm Count ◽  
Sperm Concentration ◽  
Practical Approach ◽  
Testicular Function ◽  
Special Issue ◽  
Western Countries ◽  
Meta Regression

Male infertility is a widespread condition among western countries. Meta-regression data show that sperm concentration and total sperm count have halved in the last decades. The reasons of this decline are still unclear. The evaluation of testicular function in pre-pubertal children may be effective in the timely detection of Sertoli cell (SC) disfunction, which anticipates the diagnosis of male infertility. The aim of this Special Issue is to gather together in vitro evidence on SC physiology, causes of SC dysfunction, and to suggest a practical approach to be adopted in children.

BIOLOGICALLY ACTIVE LUTEINIZING HORMONE (LH) IN PLASMA

1976 ◽  
Vol 83 (3) ◽  
pp. 454-465 ◽  
Author(s):  
P. Romaní ◽  
D. M. Robertson ◽  
E. Diczfalusy
Keyword(s):  
Menstrual Cycle ◽  
Dose Response ◽  
Gel Filtration ◽  
Biologically Active ◽  
Relative Potency ◽  
Response Curves ◽  
Plasma Pool ◽  
Dose Response Curves ◽  
Post Menopausal

ABSTRACT A recently described in vitro bioassay method for the measurement of biologically active LH (Van Damme et al. 1974) has been applied to the plasma of normally menstruating and post-menopausal women. The specificity of the procedure was established according to the following evidence: 1. Parallelism was observed between dose response curves obtained with serial dilutions of a standard LH preparation (HMG 2nd IRP) and plasma pools collected during the follicular phase, at the LH-peak, during the luteal phase and after menopause. 2. There was no evidence for the presence of any synergistic or antagonistic factor in the various plasma specimens. The assay design used to establish this consisted of assaying the standard and plasma pool separately and then together as a mixture followed by an asssessment of the difference (if any) in the potencies obtained. Strict additivity should yield a relative potency of 1.0. Plasma pools which were obtained every 2–3 days throughout the menstrual cycle were assayed using this design against the standard (HMG 2nd IRP) and against a mid-cycle plasma pool obtained at the time of the LH-peak. The latter was also assayed against partially purified plasma fractions obtained from a post-menopausal plasma pool after gel filtration and isoelectric focusing. With the exception of 3 assays, in which the estimates of relative potency were 0.91, 0.94 and 0.95, respectively, in 19 assays of additivity, the fiducial limits always included unity. 3. Non-detectable LH levels were found in the plasma or serum of either hypophysectomized or hypopituitary hypogonadal men or women treated with oestrogen/progestogen combined pills. 4. The presence of calf or human serum in the assay medium is an essential requirement for a valid comparison of standard and unknown preparations. In their absence, non-parallel dose response curves between plasma and standard were obtained. The other established criteria of reliability (sensitivity and precision) were also examined. The method is sufficiently sensitive (3.5–8.0 mIU/ml plasma; HMG (2nd IRP) as standard) for the measurement of LH throughout the cycle. The mean index of precision (λ) in 230 multiple assays was 0.040. It is concluded that the modified bioassay yields valid and reliable estimates of LH when applied to human plasma obtained throughout the menstrual cycle and after menopause.

Analysis of immunoreactive and biologically active human parathyroid hormone-peptides by high-performance-liquid-chromatography

1984 ◽  
Vol 107 (1) ◽  
pp. 60-69 ◽  
Author(s):  
T. Schettler ◽  
B. Aufm' Kolk ◽  
M.J. Atkinson ◽  
H. Radeke ◽  
C. Enters ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Parathyroid Adenoma ◽  
High Performance ◽  
Biologically Active ◽  
Peptide Sequence ◽  
Human Parathyroid Hormone ◽  
Healthy Individuals ◽  
In Vitro Bioassay

Abstract. A combination of high-performance-liquidchromatography (HPLC), sensitive radioimmunoassays and a homologous in vitro bioassay was used to characterise human parathyroid hormone (hPTH)-peptides in human parathyroid adenoma and plasma. Chromatography of several synthetic hPTH-peptides allows the calibration of the HPLC column. On the basis of sequence hydrophobicity the elution position of peptides can be predicted. A model for the determination of the minimal peptide sequence of each peptide has been developed which based on immunological and physicochemical properties allows the characterisation of unknown hPTH-peptides. Using this technique the heterogeneity of circulating hPTH-peptides in human plasma has been examined. Plasma extracts from healthy individuals, osteoporotic, hyperparathyroid and pseudohyperparathyroid patients were investigated. A uniform pattern in the heterogeneity of hPTH-peptides was detected. Using parathyroid adenoma as reference disease specific changes were characterised.

Effects of acute stimulation with luteinizing hormone-releasing hormone (LRH) on biologically active and immunoreactive serum luteinizing hormone (LH) in pubertal boys

1984 ◽  
Vol 107 (3) ◽  
pp. 289-294 ◽  
Author(s):  
Vanna Montanini ◽  
Marco Francesco Celani ◽  
Gian Franco Baraghini ◽  
Cesare Carani ◽  
Paolo Marrama
Keyword(s):  
Luteinizing Hormone ◽  
Serum Levels ◽  
Biologically Active ◽  
Acute Administration ◽  
Adult Men ◽  
Serum Luteinizing Hormone ◽  
Significant Difference ◽  
The Mean ◽  
Pubertal Boys

Abstract. The responses of biologically active LH (BIO-LH) and immunoreactive LH (RIA-LH) to acute stimulation with LRH (0.1 mg iv) were studied in 8 pubertal boys (9–15 years, 2nd–4th Tanner's stage), and in 10 healthy adult men (20–46 years). Serum levels of BIO-LH were assessed by an in vitro bioassay method based upon testosterone production by mechanically dispersed mouse Leydig cell preparations. In pubertal boys the mean BIO-LH/RIA-LH (B/I) ratio of basally secreted LH was significantly lower than in adult men (1.2 ± 0.2 (sem) and 2.2 ± 0.2 respectively, P < 0.01). After acute administration of LRH the mean B/I ratio of circulating LH showed a significant increase from the basal value in pubertal boys (2.6 ± 0.2, P < 0.01 vs basal values), whereas no significant difference in LH B/I ratios were demonstrated throughout the study period in adult men (2.1 ± 0.1, P = NS vs basal values). In agreement with this finding, the mean relative maximum response for BIO-LH (BIO-LH Δ%) was higher in pubertal boys than in adult men (1702.7 ± 500.3 and 499.6 ± 65.4% respectively, P < 0.05), whereas the mean RIA-LH Δ% was similar in both groups (609.1 ± 85.1 and 534.1 ± 75.5% respectively, P = NS). No significant differences were shown in the BIO-LH Δ area between pubertal boys (4.9 ± 0.9 area units × 103) and adult men (6.7 ± 1.2 area units × 103, P = NS), whereas the mean RIA-LH Δ area was significantly lower in the former group (1.9 ± 0.4 area units × 103 vs 3.2 ± 0.5 area units × 103, P < 0.05). Our study emphasizes that the pubertal pituitary possesses a greater responsiveness for BIO-LH than the adult pituitary, and that in pubertal boys acute stimulation with LRH evokes the release of a more bioactive form of LH.

scholarly journals Granulocytic Myeloid-Derived Suppressor Cell Exosomal Prostaglandin E2 Ameliorates Collagen-Induced Arthritis by Enhancing IL-10+ B Cells

2020 ◽  
Vol 11 ◽  
Author(s):  
Xinyu Wu ◽  
Dongwei Zhu ◽  
Jie Tian ◽  
Xinyi Tang ◽  
Hongye Guo ◽  
...  
Keyword(s):  
B Cells ◽  
Prostaglandin E2 ◽  
Plasma Cells ◽  
Suppressor Cells ◽  
Suppressor Cell ◽  
Serum Levels ◽  
Biologically Active ◽  
Collagen Induced Arthritis

The results of recent studies have shown that granulocytic-myeloid derived suppressor cells (G-MDSCs) can secrete exosomes that transport various biologically active molecules with regulatory effects on immune cells. However, their roles in autoimmune diseases such as rheumatoid arthritis remain to be further elucidated. In the present study, we investigated the influence of exosomes from G-MDSCs on the humoral immune response in murine collagen-induced arthritis (CIA). G-MDSCs exosomes-treated mice showed lower arthritis index values and decreased inflammatory cell infiltration. Treatment with G-MDSCs exosomes promoted splenic B cells to secrete IL-10 both in vivo and in vitro. In addition, a decrease in the proportion of plasma cells and follicular helper T cells was observed in drainage lymph nodes from G-MDSCs exosomes-treated mice. Moreover, lower serum levels of IgG were detected in G-MDSCs exosomes-treated mice, indicating an alteration of the humoral environment. Mechanistic studies showed that exosomal prostaglandin E2 (PGE2) produced by G-MDSCs upregulated the phosphorylation levels of GSK-3β and CREB, which play a key role in the production of IL-10+ B cells. Taken together, our findings demonstrated that G-MDSC exosomal PGE2 attenuates CIA in mice by promoting the generation of IL-10+ Breg cells.

scholarly journals A specific in vitro bioassay for measuring erythropoietin levels in human serum and plasma

Blood ◽  
1990 ◽  
Vol 76 (7) ◽  
pp. 1323-1329 ◽  
Author(s):  
AW Wognum ◽  
V Lam ◽  
R Goudsmit ◽  
G Krystal
Keyword(s):  
Human Serum ◽  
Magnetic Beads ◽  
Serum Proteins ◽  
Simple Procedure ◽  
Magnetic Bead ◽  
Biologically Active ◽  
In Vitro Bioassay ◽  
Magnetic Bead Assay

Abstract The accurate measurement of biologically active erythropoietin (Ep) in human serum and plasma using present in vivo and in vitro bioassays is difficult because of the presence of both inhibitors and non-Ep stimulators of erythropoiesis. We have developed a simple procedure to quantitatively purify Ep from serum and plasma for subsequent testing in the phenylhydrazine-treated mouse spleen cell assay. The method involves absorption of Ep to an immobilized high-affinity anti-Ep monoclonal antibody and acid elution of the antibody-bound material. After neutralization, the eluted EP is then tested directly in the in vitro bioassay without interference by other serum proteins. By using magnetic beads as a solid support for the antibody, washing and elution steps can be performed rapidly and efficiently. Recoveries of Ep after this procedure show very little sample-to-sample variation and are consistently between 45% and 55%, which is close to the maximum binding expected for the anti-Ep antibody. Coupled with the 7.4-fold concentration that this procedure affords, there is an overall increase in sensitivity of three- to fourfold, which makes this assay suitable for accurately measuring Ep levels in patients with below-average titers. Results with this magnetic bead assay indicate that accurate and reproducible estimates for Ep levels in the serum and plasma from healthy donors as well as from patients with hematologic disorders can be obtained. Titers of biologically active Ep in the sera from a group of patients with either leukemia or lymphoma were found to be elevated, and the values correlated well with titers of immunoreactive Ep measured in the Ep radioimmunoassay. Because of its specificity and high sensitivity, the magnetic bead assay is a valuable alternative to immunoassays for the measurement of elevated, normal, and even subnormal Ep levels in human serum and plasma.

scholarly journals G-CSF–stimulated Neutrophils Are a Prominent Source of Functional BLyS

2003 ◽  
Vol 197 (3) ◽  
pp. 297-302 ◽  
Author(s):  
Patrizia Scapini ◽  
Bernardetta Nardelli ◽  
Gianpaolo Nadali ◽  
Federica Calzetti ◽  
Giovanni Pizzolo ◽  
...  
Keyword(s):  
B Cell ◽  
B Lymphocyte ◽  
Serum Levels ◽  
Surface Expression ◽  
Biologically Active ◽  
Human Neutrophils ◽  
Activated Neutrophils ◽  
B Cell Homeostasis

B lymphocyte stimulator (BLyS) is a novel member of the TNF ligand superfamily that is important in B cell maturation and survival. We demonstrate that human neutrophils, after incubation with G-CSF or, less efficiently, IFNγ, express high levels of BLyS mRNA and release elevated amounts of biologically active BLyS. In contrast, surface expression of the membrane-bound BLyS was not detected in activated neutrophils. Indeed, in neutrophils, uniquely among other myeloid cells, soluble BLyS is processed intracellularly by a furin-type convertase. Worthy of note, the absolute capacity of G-CSF–stimulated neutrophils to release BLyS was similar to that of activated monocytes or dendritic cells, suggesting that neutrophils might represent an important source of BLyS. In this regard, we show that BLyS serum levels as well as neutrophil-associated BLyS are significantly enhanced after in vivo administration of G-CSF in patients. In addition, serum obtained from two of these patients induced a remarkable accumulation of neutrophil-associated BLyS in vitro. This effect was neutralized by anti–G-CSF antibodies, indicating that G-CSF, present in the serum, stimulated neutrophils to produce BLyS. Collectively, our findings suggest that neutrophils, through the production of BLyS, might play an unsuspected role in the regulation of B cell homeostasis.

Factors affecting the secretion of immunoactive inhibin into testicular interstitial fluid in rats

1988 ◽  
Vol 119 (2) ◽  
pp. 315-326 ◽  
Author(s):  
R. M. Sharpe ◽  
I. A. Swanston ◽  
I. Cooper ◽  
C. G. Tsonis ◽  
A. S. McNeilly
Keyword(s):  
Sertoli Cell ◽  
Interstitial Fluid ◽  
Positive Relationship ◽  
Cell Function ◽  
Local Heating ◽  
Serum Levels ◽  
Seminiferous Tubules ◽  
Testosterone Levels ◽  
Testicular Weight

ABSTRACT Immunoreactive inhibin was measured in testicular interstitial fluid (IF) from rats during sexual maturation or after impairment of spermatogenesis induced by ethane dimethanesulphonate (EDS), unilateral cryptorchidism or local heating (43 °C, 30 min) of the testes, to ascertain its usefulness as a marker of changing Sertoli cell function. Cultures of isolated seminiferous tubules were also studied. Inhibin was measured by a radioimmunoassay directed towards the first 26 amino acids of the N-terminus of the α-subunit, and the results confirmed for selected pools of IF by in-vitro bioassay using dispersed ovine pituitary cells. During puberty, IF levels of immunoactive inhibin fell by more than 90% (P<0·001) between 30 and 60 days of age, a decrease paralleled by the levels of androgen-binding protein (ABP), another Sertoli cell product secreted into IF. These changes also paralleled, but preceded, the fall (60%; P<0·001) in serum levels of FSH between 40 and 70 days, while the serum and IF levels of testosterone increased more than two-fold over this period. When adult rats were injected with EDS to destroy the Leydig cells, testosterone levels in IF and serum were undetectable at 3 and 7 days after treatment, were just detectable at 14 days and thereafter returned slowly towards normal by 42 days. The initial androgen withdrawal following EDS treatment caused a progressive reduction in testicular weight up to 21 days and this was accompanied by a significant increase in the serum levels of FSH and a two- to threefold increase in the IF levels of immunoactive inhibin (and also of ABP). Serum FSH and IF levels of immunoactive inhibin returned to within the normal range by 42 days when testosterone levels had normalized. In contrast, in two other experimental situations in which a marked decrease in testicular weight coupled with an increase in IF levels of ABP occurs, different results for the IF levels of immunoactive inhibin were obtained. Thus, in rats exposed to local heating of the testes, IF levels of immunoactive inhibin remained unchanged from control values at 21–40 days after treatment, a finding confirmed by bioassay results. In rats made unilaterally cryptorchid for 10 months, levels of immunoactive inhibin in IF were reduced by 60% (P<0·01) in the abdominal compared with the contralateral scrotal testis. These results suggest that (1) IF levels of immunoactive inhibin do not always change in parallel to the levels of ABP and may be a useful marker of changing Sertoli cell function, and (2) in at least two situations (puberty and after EDS treatment), there may be a positive relationship between the serum levels of FSH and the IF levels of immunoactive inhibin. This positive relationship was confirmed by in-vitro findings in which FSH and dibutyryl cyclic AMP (but not testosterone) were shown to stimulate immunoactive inhibin production by isolated rat seminiferous tubules during culture for 2–6 days. J. Endocr. (1988) 119, 315–326

In vitro bioassay for human chorionic gonadotrophin

1980 ◽  
Vol 93 (1) ◽  
pp. 114-122 ◽  
Author(s):  
T. Rabe ◽  
U. Hilgenfeldt ◽  
W. E. Merz
Keyword(s):  
Dose Response ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Lag Phase ◽  
In Vitro Tests ◽  
Intracellular Camp ◽  
In Vitro Bioassay ◽  
Assay Sensitivity ◽  
Inter Assay Variation

Abstract. The adenylate cyclase stimulation (ACS) assay is a new in vitro bioassay for human chorionic gonadotrophin (hCG) which is based on the hCG-induced accumulation of cAMP in the incubation medium of decapsulated rat testes. The detection limit for hCG is 0.7 mIU/ml (P < 0.05). A linear dose-response curve on semilogarithmic plots was obtained using 0.18, 0.45, and 1.125 IU hCG/ml. The precision of the ACS assay was satisfactory (λ-value: 0.20 + 0.02, mean ± sd), n = 14). Intra-assay variation: 15% and inter-assay variation: 20%. Medium cAMP was determined by means of a bovine adrenal protein binding assay. Sensitivity: 0.2 pmoles cAMP/ml. Range: 2 to 40 pmoles/ml. Intra-assay variations: 5% and inter-assay variation: 8%. As pre-conditions for the ACS assay, cAMP kinetics and dose-response curves were investigated. In kinetic studies of cAMP production the lag phase between hormone addition and increase of medium cAMP is shortened with higher hCG concentration. The highest concentration of cAMP was measured after an incubation of 3 h. Thereafter the concentration declines exponentially due to a decrease of intracellular cAMP formation and pre-dominating activity of extracellular phosphodiesterase. A prolongation of cAMP half-life from 16.8 min to 218 min was obtained by the addition of theophylline (70 mm). Pre-treatment of rats with 50 IU sc 48 and 24 h prior to in vitro tests caused a complete inhibition of cAMP response to hCG stimulation. Four weeks after this desensitization, the sensitivity of the testes had recovered to 80%.

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