FSH has a key role in the development and function of the reproductive system and is widely used both diagnostically and therapeutically in developmental and reproductive medicine. The accurate measurement of FSH levels, in patients for diagnosis and monitoring and in therapeutic preparations for clinical use, is essential for safe and successful treatment. Historically, FSH was defined on the basis of classical in vivo endocrine activity, and early therapeutic preparations were calibrated using in vivo bioassays. There was early recognition that reference preparations were required for calibration if the results from different laboratories were to be comparable. In response to the perceived need, the World Health Organization established the first standard for such preparations in 1959. Subsequent developments in biotechnology have led to recognition that there is no single molecule that can be uniquely defined as FSH, and that FSH can induce a range of biological activities. Several highly purified standards for FSH are now available, but discontinuity and heterogeneity of estimates of FSH activity in terms of these standards made using in vitro assays and binding assays have been noted. It is thus essential that any measurement of FSH include specification both of the standard with which the measured FSH is compared and the assay method used for that comparison.
The role of arachidonic acid in the regulation of steroidogenesis in rat Leydig cells was studied. A dose- and time-dependent biphasic effect on maximal and submaximal LH- and dibutyryl-cAMP-stimulated testosterone production was found. The locus of the inhibition, which occurred during 3 h incubation, was prior to the side chain cleavage of cholesterol and after cAMP production. The same inhibitory effect was found with the protein kinase C (PKC) activators, phorbol-12-myristate, 13-acetate (PMA) and oleic acid, also with no change in LH-stimulated cAMP production. Arachidonic acid, PMA, and diolein, all stimulated PKC activity in a dose-dependent fashion in partially purified Leydig cell homogenates. When the cells were incubated for 5 h, arachidonic acid potentiated LH- and dibutyryl-cAMP-stimulated testosterone production. Similarly, incubation with PMA for 5 h, potentiated subsequent basal and dibutyryl-cAMP-stimulated testosterone production. PKC was down-regulated over 5 h (but not during 3 h) by pretreating Leydig cells with PMA or arachidonic acid in the presence of LH. Lipoxygenase and cyclooxygenase inhibitors did not alter the stimulatory effects of arachidonic acid. We conclude that the short-term inhibitory effect of arachidonic acid (and PMA) is via activation of PKC, but when protein kinase C (PKC) is down-regulated by these ligands, steroidogenesis is enhanced. These results suggest that steroidogenesis is normally under tonic inhibitory control by PKC.
Since the 1950s, serological diagnosis of ovine enzootic abortion (OEA), caused by strains of Chlamydia psittaci, has been based mainly on the complement fixation test (CFT), which is neither particularly sensitive nor specific since antibodies to other chlamydial and enterobacterial pathogens may be detected. In this study, a recombinant enzyme-linked immunosorbent assay (rELISA) (medac, Hamburg, Germany), based on a unique chlamydial genus-specific epitope of Chlamydia trachomatis L2 lipopolysaccharide, was evaluated for sensitivity and specificity as a primary screening assay for OEA by comparison with the CFT. A comparative inclusion immunofluorescence assay (IFA), in which antibody titers to C. psittaci and Chlamydia pecorum were examined, was used as the reference test for 573 serum samples from four flocks. Reactivity to C. pecorum was measured since inapparent intestinal infections by C. pecorum are believed to be common in British flocks. In detecting positive sera from an abortion-affected flock, in which a C. pecorum infection was also suggested by IFA, the rELISA outperformed the CFT with significant evidence for increased sensitivity (P ؍ 0.003). In two flocks in which C. pecorum infections alone were suggested by IFA, the rELISA and CFT were prone to detect low levels of false-positive results, but the values were not significant. The rELISA provided results in one flock in which sera that were anticomplementary could not be resolved by the CFT. In another flock in which abortion had not occurred but infection by both chlamydial species was suspected, no significant difference was found between the sensitivities of the rELISA and CFT. The rELISA could not differentiate ovine C. psittaci and C. pecorum infections but was shown to be a more sensitive primary screening test for OEA than was the CFT, particularly where abortion had occurred and even when antibodies due to additional inapparent infection(s) by C. pecorum were present.
Background: Prostate-specific antigen (PSA) measurements in serum by immunoassay are widely used in the screening, diagnosis, and monitoring of patients with prostate cancer although the lack of common reference reagents has led in the past to wide differences in estimates. We report here the results of a WHO international collaborative study in which two preparations of PSA representative of the main immunoreactive components in serum, free PSA and PSA 90:10, and a preparation of recombinant DNA-derived PSA were assessed as potential standards for the calibration of diagnostic immunoassays for PSA.
Methods: Coded vials of the candidate materials and serum preparations containing PSA in the clinically important range were provided to the 10 laboratories in the study, and participants were asked to perform PSA assays currently in use in their laboratories. Data from 89 immunoassays by 26 different method-laboratory combinations were contributed to the study and analyzed centrally at the National Institute for Biological Standards and Control.
Results: Potency estimates of the preparations relative to the in-house calibrators were in good agreement with the target value of 1 μg of total PSA/vial, the preparation of free PSA giving 1.10 μg/vial (95% confidence interval, 0.99–1.21 μg/vial) and PSA 90:10, 1.11 μg/vial (95% confidence interval, 1.04–1.18 μg/vial). No immunoreactivity was detected in ampoules containing the recombinant material. Use of a common standard of PSA 90:10 significantly reduced the between-laboratory geometric coefficients of variation for serum samples included in the study and gave a much narrower range of potency estimates.
Conclusions: The preparation of free PSA was established by WHO as the First International Standard for PSA (free) with an assigned content of 1 μg of total PSA per vial. In addition, the preparation of bound PSA was established as the First International Standard for PSA (90:10) with an assigned content of 1 μg of total PSA per vial.
The effect of anti-inhibin antibodies on gonadotrophin surge attenuating/inhibiting factor (GnSAF/GnSIF) and its effect on gonadotrophin secretion in a pituitary cell bioassay were determined by culturing rat pituitary cells in a serum-free medium to which inhibin and a partially purified preparation of gonadotrophin surge inhibiting factor (GnSIF) were added. The samples were treated with an anti-inhibin antiserum and basal and GnRH-stimulated FSH and LH secretion were measured by radioimmunoassay of the culture supernatant. Inhibin had a dose-dependent inhibitory effect on both basal and GnRH-stimulated FSH secretion and also on GnRH-stimulated LH secretion. Anti-inhibin antibody blocked the inhibition of FSH secretion, except at the highest two doses of human inhibin (1.25 ng ml-1, P = 0.04; 2.5 ng ml-1, P = 0.0004). The GnSIF preparation inhibited basal and GnRH-stimulated FSH and GnRH-stimulated LH secretion and was not affected by the anti-inhibin antibody. The novel hormone, GnSIF, is different from inhibin.
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