An improved in vitro bioassay for follicle-stimulating hormone (FSH): suitable for measurement of FSH in unextracted human serum

FSH bioactivity was measured by means of FSH-dependent aromatase activity (conversion of androgen substrate to estradiol). Assay sensitivity was optimized by the use of immature (7-10 days old) rats as Sertoli cell donors, serum-free medium for incubation, phosphodiesterase inhibitor (methylisobutylxanthinine), serial dilution of FSH in medium containing 1% BSA, delayed addition of FSH for 72 h after cell plating, and 19-hydroxyandrostenedione (2.5 X 10(-6) M) as the aromatizable androgen substrate. The method consisted of subjecting the decapsulated immature rat testes to a 2-step collagenase dispersion, plating the cells in medium [Dulbecco's Modified Eagle's Medium-Ham's F-10 (1:1)] containing growth factors and methylisobutylxanthinine for 72 h, adding increasing doses of FSH to the standard curve or small volumes of serum to the test vials as well as 19-hydroxyandrostenedione for 24 h, and measuring estradiol by RIA in dilutions of the medium. Using NIAMDD human (h) FSH-2 as the bioassay standard, the useful range of the assay was 0.01-5.0 ng/ml. Specificity was determined by the addition of graded doses of hLH, hTSH, ACTH, hGH, hPRL, and hCG. The minor degree of FSH bioactivity observed in a few hormone preparations was accounted for by the degree of FSH contamination in them. Mean intra- and interassay coefficients of variation were 9% and 11%, and the index of precision was 0.049. This bioassay was used to determine the bioactive FSH content of pituitary extracts, tissue culture media, elutions from columns, and isoelectrically focused samples. More importantly, small quantities of human sera gave responses parallel to the standard curve in a minimum of two dilutions. The bio- to immunoreactive ratios, expressed as the mean +/- SEM (NIAMDD-hFSH-2), were 0.66 +/- 0.2 in boys (n = 6), 0.78 +/- 0.2 in pubertal girls (n = 6), 1.18 +/- 0.2 in men (n = 13), 1.24 +/- 0.1 in postmenopausal women (n = 30), 1.94 +/- 0.3 in the follicular phase (n = 19), 6.2 +/- 1.4 in the ovulatory phase (n = 19), and 1.6 +/- 0.4 in the luteal phase (n = 19) of the normal menstrual cycle. These results indicate that the bio- to immunoreactive hFSH ratio in the circulation, is dependent upon the hormonal milieu of the subject.     

An evaluation of factors affecting the in vitro bioassay for erythropoietin.

Two main aspects of the in vitro mouse foetal liver cell assay for Erythroid Stimulating Factor (ESF) in human sera have been investigated. The haem extraction process has been shown to give specific and quantitative recovery of 59Fe labelled haem from haemoglobin thus confirming that the material assayed in human sera is stimulating the synthesis of this protein. The extraction procedure can be simplified considerably by prior mixing of the reagents without significantly influencing the results. Several serum constituents (citrate, testosterone, B12, folic acid and iron) have been investigated over a range of concentrations for possible effects on the cultures. Generally only small effects on haem synthesis were observed. It is concluded that variations in the levels of these factors in sera from treated patients will not produce any significant alterations in the estimated ESF concentrations.     

An improved animal model for studying desferrioxamine

A hamster model has been developed for studying desferrioxamine. The hamster shows many similarities to man in terms of plasma stability and metabolites formed from desferrioxamine. The [⁵⁹Fe]ferritin derived from rats has been shown to be sequestered into liver parenchymal cells when injected intravenously into hamsters. The technique has proved sufficiently sensitive to enable detection of differences of < 1% of the radioactivity administered in the elimination of iron. Alterations in iron excretion were seen when dosing desferrioxamine via different routes. The principal route of iron excretion was into the intestines. The effectiveness of the dosing routes for desferrioxamine in removing iron were subcutaneous (10.5%) > intravenous (6.25%) > oral (3.66%) > control (2.19%). A dose–response relationship was demonstrated using the intravenous dose route. The model offers a simple method for comparing the efficacy of administration routes for determining the optimal use of desferrioxamine.     

An improved method for lupus anticoagulant detection

The diagnosis of lupus anticoagulant (LA) becomes difficult when there is a weak titer of LA antibodies. This study demonstrates that 20 minutes' incubation increases the sensitivity of the mixing test to diagnose LA by 23% and to suspect the antibodies by 66%.     

An improved serodiagnostic procedure for visceral leishmaniasis

The enzyme-linked immunosorbent assay (ELISA) is a sensitive and specific serodiagnostic method for leishmaniasis. In this report, we describe how this versatile assay can be improved by the use of protein A or protein G conjugates for the specific detection of Leishmania antibody in the sera of patients with visceral leishmaniasis. In direct comparisons with anti-immunoglobulin conjugate, enzyme-linked protein A gave significantly higher absorbance values for positive sera without a corresponding increase in absorbance values for sera from normal individuals or from patients with other diseases known to cross-react with leishmaniasis. The effect was to increase the distance between positive and negative values, which aided in the interpretation of the results. This also permitted visual distinction between positive sera and negative or weakly reactive sera. The assay was effective using either blood or serum as the source of primary antibody. A further advantage of protein A over anti-Ig conjugate was its ability to detect specific antibody in dog as well as human sera. Finally, we demonstrated the usefulness of the protein A ELISA with a recombinant leishmania antigen, gp63.