F. Dray

Radioimmunoassay of Prostaglandins Fα, E1 and E2 in Human Plasma

Abstract. Antibodies against prostaglandins (PG)F2α, E1 and E2 were obtained in rabbits immunized with respectively PG F2α, PG E1 and PG E2 conjugated to bovine serum albumin by carbodiimide. A radioimmunoassay capable of measuring 7 pg of PG Fα, 2 pg of PG E2 and 14 pg of PG E1 in human peripheral plasma is described. Plasma samples (pH 3, citric acid) are extracted with cyclohexane: ethyl acetate, 1:1 and then chromatographed on silicic acid columns to separate the prostaglandins into three fractions: fraction I, PG A, PG B and some unknown immunoreactive compounds; fraction II, PG E and fraction III, PG Fα. The recovery is 80 % ± 6.2. Mean plasma levels in adults of PG Fα and PG E, expressed in pg/ml: ‐PG Fα 12 ± 2.8 (n = 25 men), 8 ± 2.3 (n = 18 women, follicular phase), 7 ± 1.4 (n= 18 women, luteal phase). ‐PG El 40.5 ± 7.6 (n = 13 men), 38 ± 17.1 (n = 10 women). ‐PG E2 4.5 ± 1 (n = 12 adult subjects).

Radioimmunoassay of Prostaglandins Fα, E1and E2in Human Plasma

Abstract. Antibodies against prostaglandins (PG)F2α, E1 and E2 were obtained in rabbits immunized with respectively PG F2α, PG E1 and PG E2 conjugated to bovine serum albumin by carbodiimide. A radioimmunoassay capable of measuring 7 pg of PG Fα, 2 pg of PG E2 and 14 pg of PG Ej in human peripheral plasma is described. Plasma samples (pH 3, citric acid) are extracted with cyclohexane: ethyl acetate, 1:1 and then chromatographed on silicic acid columns to separate the prostaglandins into three fractions: fraction I, PG A, PG B and some unknown immunoraactive compounds; fraction II, PG E and fraction III PG Fα. The recovery is 80 %± 6. 2. Mean plasma levels iu adults of PG Fa and PG E, expressed in pg/ml: ‐PG Fα 12 ± 2. 8 (n = 25 men), 8 ± 2. 3 (n = 18 women, follicular phase), 7 ± 1. 4 (n = 18 women, luteal phase). ‐PG E1 40. 5 + 7. 6 (n = 13 men), 38 + 17. 1 (n = 10 women). ‐PG E2 4. 5 ± 1 (n = 12 adult subjects).

RADIOIMMUNOASSAY OF METHIONINE‐AND LEUCINE‐ENKEPHALINS IN REGIONS OF RAT BRAIN AND COMPARISON WITH ENDORPHINS ESTIMATED BY A RADIORECEPTOR ASSAY

Abstract— Radioimmunoassays (RIAs) selective for methionine‐enkephalin (Met‐ENK) and leucineenkephalin (Leu‐ENK) have been developed using competition towards binding of 10 pM 125I‐enkephalins to antibodies raised in rabbits against ENKs coupled to ovalbumin with carbodiimide. The high sensitivity of the RIAs (IC50 0.57 nm and 0.55 nm for Met‐ and Leu‐ENK, respectively) allowed estimation of the enkephalin content in extracts of all rat brain regions. Regional levels are compared with those determined on the same extracts by a radioreceptor assay (RRA) using competition towards binding of 5 nm [3H]Leu‐ENK to rat striatal membranes. Optimal conditions for killing the animals and extracting the endorphins have been carefully investigated: killing by rapid microwave irradiation was not found necessary as long as brain regions were homogenized into 0.1 n‐HCl before deproteinization.

Analytical methods for thromboxane B2 measurement and validation of radioimmunoassay by gas liquid chromatography-mass spectrometry

A sensitive and specific radioimmunoassay using a 125I tracer of high specific radioactivity was developed for thromboxane B2 and was applied to the determination of the in vitro biosynthesis of this compound in some systems (i.e. human washed platelets, human platelet rich plasma (PRP) and rat spleen homogenates). The assays were also evaluated by mass spectrometry; levels measured by these two analytical methods were very similar. The results obtained for washed human platelets with a thin layer radiochromatographic method were in good agreement with the two preceeding methods.

125I derivatives of prostaglandins: A novel approach in prostaglandin analysis by radioimmunoassay

We report the production of radioactive iodinated (125 I) derivatives of prostaglandins E1, E2, F2alpha and their use in radioimmunological assays. Histamine or tyramine was coupled to the prostaglandins carboxyl group and the iodination was accomplished using the chloramine T method. The high specific radioactivity of these tracers and the resolution of the purification procedure allowed the detection of 0.5 pg of prostaglandins. A comparison with tritiated prostaglandin was made and showed a 10-fold gain in sensitivity. Furthermore in the case of the prostaglandin E1 system using 125I-labelled histamine or tyramine as tracer the cross reaction curves obtained were different from those obtained with [3H]prostaglandin E1; we suggest that the blocking of the carboxyl group alters the prostaglandin E1 structure, modifying its immunoreactivity.

Evidence for the presence of enkephalin in catecholaminergic neurones of cat locus coeruleus

The distribution of enkephalin (Enk) and tyrosine hydroxylase (TH) immunoreactivity in the cat locus coeruleus (LC) has been studied with indirect immunofluorescence technique. After intratissular injection of colchicine numerous enkephalin-containing cell bodies were seen throughout the rostrocaudal extent of the LC complex. Comparison of 8 micrometers-thick consecutive sections treated with antiserum to Enk of TH, a specific marker for catecholaminergic neurons, shows that most cells containing TH also present Enk immunoreactivity. This study provides the first evidence for the coexistence of enkephalin and catecholamine in CNS neurons.

Specific binding of [3H]prostaglandin E2 to rat brain membranes and synaptosomes.

There is saturable, reversible and specific binding for [3H]prostaglandin E2 (PGE2) to rat brain membranes. This binding is of high affinity, selectively distributed with a maximum in the hypothalamus, the amygdala and the posterior pituitary, and is associated subcellularly with the synaptosomal fraction. This specific PGE2 binding has the characteristics expected for receptors, so opening new perspectives which might clarify the role of PGs in the brain.

Haemolymph levels of juvenile hormone, ecdysteroids and protein during the last two larval instars of Locusta migratoria

Abstract The radioimmunoassay of ecdysteroids and juvenile hormone has enabled us to relate hormone levels to haemolymph protein concentrations and weight increase during the 4th and 5th instar of the migratory locust. The two hormones are never present in high concentrations in the blood simultaneously. The levels of ecdysteroids are high on the 5th day during the 4th larval stage: they show a small peak on the 3rd day, and then a large peak on the 8th day during the 5th instar. JHI-immunoreactive substances are high during the first 4 days of the 4th instar, and during the first 5 hr during the 5th instar. Protein concentrations in the haemolymph begin to rise when ecdysteroid levels increase during stage 4, and immediately after the small peak (on day 3) in the 4th stage larva. The rise in protein levels is correlated with an increase in weight.

Enzyme immunoassay of progesterone at the picogram level using β-galactosidase as label

We report here the first sensitive enzyme immunoassay of a hapten. A progesterone beta galactosidase conjugate was prepared using carbodiimide as a bifunctional reagent. Rabbit progesterone antisera were previously obtained. The separation of the bound from the free fraction of the label was performed with the help of polymerized anti rabbit gamma-globulins. The enzyme activity of the bound fraction was determined with O-nitrophenyl-beta-D-galactoside as substrate. Specificity and sensitivity (approximately 15 pg) of this enzyme immunoassay can be successfully compared with radioimmunoassay performances. It thus provides a non radioactive, inexpensive and reliable method of small molecule quantitation.

Radioimmunoassay of arthropod moulting hormone: ß-ecdysone antibodies production and 125i-iodinated tracer preparation

Ecdysones (or moulting hormones) are polyhydroxysteroids which are involved in ecdysis of arthropods. Since the identification of two hormone forms (cr and /3) many authors have described the chemistry and metabolism of these compounds [ 11. Biological [2], physicochemical [3-61 and radioimmunological [3,7,8] tests have also been developed for their titration. No direct data have yet been reported regarding the precise localization of intracellular sites of production and storage of ecdysones. An immunocytochemical study seemed to us particularly appropriate; its success depends on obtaining, essentially for ultrastructural studies, specific antibodies with high binding parameters. We report here the conditions of anti ecdysone antibody production and the radioimmunoassay developed with an iodinated derivative as tracer.