Quality control of coated antibodies: new, rapid determination of binding affinity.

A procedure is described for the determination of the affinity constant between a fluid-phase biotinylated antigen and a solid-phase monoclonal antibody. This procedure allows evaluation of the efficiency of an antibody as a coated tool for an immunoassay. For this purpose, the biotinylation of the antigen and its further quantitative measurement by streptavidin-peroxidase led to a single reversible interaction, the binding affinity of which greatly determines the quality of the assay. The free and bound fractions of the biotinylated antigen were obtained in wells coated with a low level of immobilized antibodies. At the equilibrium state, the free antigen present in the supernatant of these wells was further transferred to high level antibody coated wells which captured all the free antigen molecules. These molecules were quantified using a standard curve established with known concentrations of biotinylated antigen, also incubated in wells coated with the high level of antibody.

Long-term results of growth hormone treatment in France in children of short stature: population, register based study.

OBJECTIVES: To describe the growth of children treated with growth hormone and to evaluate the prognostic factors for height at the end of treatment. DESIGN: Register based cohort study. SETTING: French national register of all children treated with growth hormone. SUBJECTS: 3233 short stature children (3165 of whom were deficient in growth hormone) who were treated with growth hormone (excluding children with Turner's syndrome) and whose treatment started between 1973 and 1989, last data being recorded in December 1993. MAIN OUTCOME MEASURES: Annual changes in height, and height at the end of treatment. RESULTS: Mean height SD score at the end of treatment, after a mean of 4.3 years, was -2, corresponding to gain in mean height SD score of 1 and to a height SD score of 1.1 below target height. In all, 923 children prematurely stopped taking growth hormone treatment, mainly because of insufficient response (insufficient growth) or tiredness. Variables that predicted height at the end of treatment were age, target height, aetiology of short stature, use of puberty inhibitors, and type of growth hormone. CONCLUSIONS: The outcome of children of short stature with growth hormone deficiency who were treated with growth hormone has been less favourable than initially assumed. Growth hormone treatment has not restored normal growth to these children. The highly demanding nature and high costs of this treatment require an optimised prescription, and this remains to be determined.

Growth hormone testing for the diagnosis of growth hormone deficiency in childhood: a population register-based study.

Evaluation of GH secretion using pharmacological GH stimulation tests (GHST) remains a current practice, although the reliability of GHST has been questioned, and many pitfalls have been pointed out. We have analyzed all of the 6373 GH stimulation tests that led to the initiation of GH therapy in 3233 children treated in France from 1973-1989. Tests and GH measurements were performed by individual centers and collected by the Association France-Hypophyse. GH deficiency (GHD) was due to craniospinal irradiation (11%), was due to organic causes or associated with multiple deficiencies (22%), or was considered idiopathic (65%); 2% of the patients were considered non-GHD. Eleven different pharmacological tests were used, and 62 of the 66 theoretical pairs of tests were used at least once. The most frequent combination of tests (ornithine in one instance and insulin in another) was used in 12.7% of patients. The reliability of the GH peak measured by comparing the results of 2 tests in the same patient was poor, as measured by intraclass correlation coefficients below 0.8. Multivariate analysis identified several parameters positively or negatively associated with peak plasma GH: calendar year of initiation of treatment, etiology of GHD, height SD score, bone age SD score, puberty, weight SD score, genetic target height SD score, and the nature of the pharmacological agent used. We believe that several of these factors (weight SD score, genetic target height SD score, and nature of the agent) identify biases in the diagnosis of GHD. We conclude that GHST should be performed with a very limited number of agents, interpreted after the establishment of reference values in age-matched normal children, and associated with other clinical and biochemical parameters for establishing the diagnosis of GHD.

Comparative activity of peroxidase-antibody conjugates with periodate and glutaraldehyde coupling according to an enzyme immunoassay.

Horseradish peroxidase is often used as an antibody-coupled enzyme and several procedures have been developed to obtain IgG-peroxidase conjugates. The most widely used are coupling with periodate or glutaraldehyde. To compare the efficiency of these methods, the authors conducted periodate coupling or glutaraldehyde coupling in one or two steps, using the same batches of peroxidase, C-reactive protein (CRP) and anti-CRP monoclonal antibodies to develop a specially sensitive Elisa for CRP. Comparison of immunoenzymatic activities showed that periodate-mediated conjugation was much more efficient, because the activity of the coupling products was about 100 times greater than that of the products obtained after one or two-step conjugation with glutaraldehyde. The lower coupling efficiency observed with glutaraldehyde was not due to inactivation of the coupling agent or to a possible decrease in the affinity of the conjugates for CRP due to the coupling procedure. The differences in efficiency can be ascribed to the fact that periodate induced more coupling sites than glutaraldehyde. Periodate is therefore a better coupling agent for preparing conjugates to be used in Elisa or related techniques, in which conjugate size does not hinder accessibility to the antigen.

[Ovulation inhibition with medrogestone administered on the 4th through the 24th day of the cycle]. / Inhibition de l'ovulation par la médrogestone administrée du 4e au 24e jour du cycle.

Medrogestone (M) is a derivative of 17 methylprogesterone (P) used for P insufficiency at oral doses of 5 to 15 mg/day (D). We studied the ability of M to inhibit cyclic pituitary-ovarian activity when given at a dose of 10 mg/d from D4 to D24. Ten healthy Caucasian females, aged from 21 to 33, volunteered for an open 2 consecutive cycle study (cycle 1 = control, cycle 2 = M). At inclusion mean (+/- SD) cycle length was 28.6 +/- 1.9 D. Plasma LH, FSH, E2, P, were measured daily from D10 to D20 and at D22, 24, 26. During cycle 1, every subject showed an ovulatory pattern with mid cycle E2 peak (151-400 pg/ml), LH peak (12-59 mUI/ml) and luteal P rise (9.4-22.8 ng/ml). Under M ovulatory surges were suppressed in each of the 10 subjects and P remained below 0.8 ng/ml. These data show that in addition to its known progestomimetic effect, M is a potent ovulation inhibitor when given from D4 to D24 of the cycle.

[Plasma concentration of C-reactive protein in patients with high estrogen levels]. / Concentration plasmatique de la protéine réactive C chez des patientes en situation de forte imprégnation estrogénique.

The monitoring of inflammatory activity in patients with a high level of estrogen is controversial because the significance of a raised estradiol level on C-reactive protein (CRP) concentrations is a debated question. This prompted us to assay CRP by a sensitive Elisa in a sample of 30 patients with ovarian stimulation for in vitro fertilization, thus with high levels of estradiol. For 15 of these women, six to nine plasma samples were analyzed allowing a kinetic study of plasma levels of CRP, estradiol and sex steroid-binding plasma protein (SBP). No significant correlation was found between the concentrations of estradiol and CRP for the 30 patients. In the kinetic study, as mean estradiol levels rose exponentially from 50 to 1400 ng/l between day 5 and 14, the CRP level tended to vary markedly from one patient to another and sometimes from day to day, but there was never any relation with estradiol level. Furthermore, CRP did not significantly modify the slope of the regression line between estradiol concentration and the day of the menstrual cycle. In contrast, the effect of estradiol on SBP was clear, which supports the absence of estradiol effect on CRP level.

Ligand-binding studies with a 23 kDa protein purified from bovine brain cytosol.

Ligand-binding studies were performed with a basic 23 kDa protein purified from bovine brain cytosol. By equilibrium dialysis experiments bromosulfophthalein, dehydroepiandrosterone sulfate and oestradiol-17 beta were demonstrated to bind to the protein with association constants of 1 X 10(6), 1 X 10(4) and 1 X 10(3) l/mol, respectively. Indocyanine green, Evans blue and Rose Bengal were not bound. The protein was further characterized as a phosphatidylethanolamine-binding protein, while phospholipid transfer assays proved negative. The so far investigated binding characteristics of the 23 kDa cytosolic protein, together with previously demonstrated sequence homologies with other known cytosolic proteins, suggest its involvement in lipid metabolism.

[De-etherification of estradiol 3-propyl ether by subcellular fractions of rat liver]. / Etude de la dééthérification du 3-popyl éther d'estradiol par les fractions subcellulaires de foie de rat.

The incubation of PE2 ((6,7-3H)-labelled 3-propyl ether of estra-1,3,5(10)triene-3, 17 beta-diol (estradiol)) with various subcellular fractions of rat liver indicated that the hepatic metabolism of this compound occurs mainly in the microsomal fraction. In addition to the formation of 3-propyl ethers of estra-1,3,5(10)triene-3-ol-17-one (estrone) and estra-1,3,5(10-triene-3, 16alpha, 17 beta-triol (estriol) directly deriving from PE2, the microsomal proteins carried out the deetherification of the propyl ether group leading to phenolic steroids; among them, estradiol, estrone, and estriol were characterized. Protein-bound and water-soluble metabolites were found; the effects of glutathione and of the incubation conditions were in agreement with the thioconjugation of these derivatives. The microsomal metabolism of PE2, and specially the deetherification reaction, required the presence of oxygen and of NADPH as cofactor, the optimum pH ranging from 7.4 to 8. The participation of cytochrome P450 in these metabolic pathways was shown by a partially inhibited catabolism with carbon monoxide and by a more active metabolism in males than in females and when animals were pretreated with phenobarbital. These results allowed us to conclude that the hepatic deetherification of PE2 is carried out by a microsomal oxidative system which is very similar to the system involved in the demethylation of methyl ethers of estrogens.

[Kinetics of transcutaneous penetration and fixation of 3-propoxy-17-methoxyestradiol and estradiol in various rat tissues]. / Cinétiques de pénétration transcutanée et de fixation du 3-propyl éther, 17-méthyl éther oestradiol et de l'oestradiol au niveau de différents tissus chez la ratte.

The transcutaneous penetration of 3-propyl ether, 17-methyl ether oestradiol (POM) occurs by a diffusion phenomenon and does not seem to be modulated by a cutaneous receptor as it is the case for oestradiol. After transcutaneous administration of POM and oestradiol, a comparison of the kinetics of uptake on the uterus and of uterotrophic effects, as well as an analysis of radioactivity taken up by a partition method between petroleum ether and sodium hydroxide, indicates that cleavage of both ether groups of POM occurs leading to estradiol. It is likely that this de-etherification takes place in the liver after a period of quiescence. The lipophilic nature of POM allows an obvious uptake by the aorta and a very significant uptake by the adipose tissue. The etherification of the alcohol functions of oestradiol allows an adequate protection of the hormone against hepatic catabolism. This may explain, along with the release of metabolites taken up by the adipose tissue, that POM is bound to a greater extent than oestradiol by various tissues.

[Experimental data on estradiol 3,17-diethers (author's transl)]. / Recherches expérimentales sur les 3, 17-diethers de l'estradiol.

Estradiol ethers reveal a biological activity in female rats which decreases according to the length of the side chain at position 3. Further, percutaneous injections have an activity which is 6 to 30 times greater that subcutaneous ones. This suggests that de-etherification could occur, leading to estradiol. After percutaneous administration of tritiated promestriene (3-propyl ether, 17-methyl ether estradiol) and estradiol, a comparison of the uptake on the uterus and of uterotrophic effects as well as an analysis of radioactivity taken up indicates that a cleavage of both ether groups of promestriene occurs leading to estradiol. This de-etherification takes place in the liver, as demonstrated by perfused liver experiments. With promestriene, antagonist activity is shown on the seborrheic androgen-stimulated rat and on 5-alpha-reductase activity in vitro and in vivo.