Differences in circulating concentrations of total, free and bound leptin relate to gender and body composition in adult humans.

We describe a radioimmunoassay (RIA) for total leptin and a gel filtration procedure for the separation of free and bound leptin in human serum. The RIA, based on a locally prepared antibody, has a minimum detection limit of 0.9 ng/mL, a working range (CV < 10%) of 2.5-50 ng/mL, inter-assay precision of 10.2, 7.2 and 8.9%CV at 7.9, 15.4 and 30.0 ng/mL, respectively, 94% recovery of exogenous leptin (range 81.1-120.6%), exhibited parallelism and demonstrated no significant cross-reactivity or interferences. A difference plot of results from this method and those from a commercially available kit (Linco Research) demonstrated satisfactory agreement up to concentrations of 50 ng/mL total leptin, with no significant bias. A gender-dependent correlation was obtained between body mass index (BMI) and total leptin (r = 0.91, P<0.001, n = 75 for men; r = 0.79, P<0.001, n = 72 for women), with women having higher leptin concentrations than men for any given BMI. Gel filtration studies (inter-assay precision: 4.7%CV, n = 18) demonstrated that a variable fraction (between 10% and 40%) of total leptin in serum was bound with high affinity (Keq = 1.0-1.45 x 10(9) L/mol) to a non-albumin, non-lipid macromolecule. Binding affinities were found to be similar irrespective of gender or fat mass. A significant positive correlation between free or bound leptin concentrations and BMI was obtained for both men and women (r = 0.87-0.94); free and bound leptin concentrations were also significantly higher in women (P<0.01) than in men for any given BMI, and higher in obese (P<0.01) than in lean individuals. We conclude that leptin 'resistance' associated with obesity cannot be accounted for by reduced free leptin concentrations in serum and that the methods described are suitable for the investigation of total, free and bound leptin for both clinical and research purposes.

Development and evaluation of a direct immunofluorimetric assay for urinary growth hormone.

We describe a two-site immunofluorimetric assay for urinary growth hormone that is resistant to interference from a wide range of urinary constituents and therefore eliminates the need for sample pre-treatment. A microtitre plate format is used with specific orientation of capture antibody on a polystyrene surface carrying a hydrazide group. Europium-labelled F(ab)2 is the fluorophore and time-resolved fluorimetry with co-fluorescence enhancement the signal detection system. Inter-assay precision was 11.3% at 5.2 ng/L and 10.3% at 44.3 ng/L, minimum detection limit (22% coefficient of variation, CV) was < 1.0 ng/L, working range (< 10% CV) was 0-100 ng/L and quantitative recovery and good parallelism were demonstrated. This convenient and sensitive assay is suitable for the routine measurement of human growth hormone (hGH) in urine.

Specificity of two-site immunometric assays.

The assessment of specificity in two-site immunometric assays is complex. Conventional approaches based on standard substitution with cross-reactant may yield misleading results. An 'in-house' immunoradiometric assay (IRMA) for human growth hormone (hGH) appeared free from cross-reactivity with human placental lactogen (hPL) in a single point standard substitution experiment performed by the United Kingdom External Quality Assessment Scheme (UK EQAS). However, detailed studies based on the substitution of hGH standards with hPL cross-reactant, the addition of hPL cross-reactant to hGH standards and the recovery of added hGH from endogenous cross-reactant (sera from pregnant subjects) revealed gross cross-reactivity of hPL in the hGH assay. We recommend that the validation of assay specificity should require the demonstration of quantitative recovery of a range of analyte concentrations from human sera that contain varying endogenous concentrations of cross-reactant.

Application of solid-phase antibodies to radioimmunoassay. Improved performance of Dynospheres following affinity purification of antisera.

Affinity purification of antibody before covalent binding to pre-activated micro-particles (Dynospheres) was necessary to overcome the reduced coupling capacity evident with low titre antisera, particularly antihapten sera. Optimally prepared Dynospheres anti-thyroxine provided a radioimmunoassay with good performance and economical usage of microparticles. Assay sensitivity was less than 10 nmol/l while precision across the clinically relevant dose range (50-150 nmol/l) was 3% CV using 125 micrograms particles per assay tube.

How sensitive are immunometric assays for thyrotropin?

The usual method for calculation of the "sensitivity" of thyrotropin immunometric assays is multireplicate analysis of the zero analyte standard. Although this is a statistically valid estimate of the scatter likely to be found in the response variable, it is unrelated to normal analytical practice (usually analysis in duplicate) and estimates intra-assay errors only. This study was designed to assess the analytical performance of 10 immunometric assays used routinely for measurement of thyrotropin in human serum. Response data from each assay were accumulated to provide (a) an estimate of "sensitivity" from multireplicate analysis and (b) an estimate of "minimum detection limit," relating directly to errors associated with routine performance and derived from a minimum of 500 duplicate analyses. We conclude that the "minimum detection limit" should be promoted as a more meaningful measure of assay performance at low analyte concentrations than the "sensitivity" derived from multireplicate analysis of the zero-analyte standard.

Performance of the two-site immunoradiometric assay for serum thyroid-stimulating hormone. Effects of changes in solid-phase matrix and antibody coupling chemistry.

Ten solid-phases were evaluated for their usefulness in a two-site immunoradiometric assay for serum thyroid-stimulating hormone. The criteria used to assess each reagent included the minimum detection limit attainable, the change of binding over the concentration range (signal: noise ratio), and ease of preparation of the reagents. Of all the solid phases tested, Sepharose CL-4B and activated CH-Sepharose 4B showed the characteristics best suited to IRMA, while epoxy-activated Sepharose 6B and Ultrogel ACA-44 were marginally less effective.

Circadian variation of thyrotrophin, determined by ultrasensitive immunoradiometric assay, and the effect of low dose nocturnal dopamine infusion.

The effect of low dose dopamine infusion on the circadian rhythm of thyrotrophin (TSH), prolactin and cortisol in a group of six healthy male volunteers is reported. Subjects were infused in random order with either saline (154 mmol/l NaCl solution; control) or dopamine (0.1 and 1 microgram min-1 kg-1) between 21.00 and 01.00 hours, in random order. The serum TSH profile was characterized by a maximal peak occurring at 23.00 hours and higher nocturnal than diurnal values. Superimposed on this are short term oscillations in serum TSH levels, typical of an ultradian rhythm. The maximal peak in TSH, occurring at 23.00 hours, was abolished by dopamine infused at a rate of 1 microgram min-1 kg-1, and was unaffected by the lower rate of dopamine infusion (0.1 microgram min-1 kg-1). The serum prolactin profile was characterized by a peak occurring soon after the onset of sleep (23.30-00.30 hours), which fell during the morning, and began to rise in late evening. Low dose dopamine (0.1 microgram min-1 kg-1) had a slight but insignificant effect with decreased prolactin levels at the end of the infusion whereas the higher dopamine dose was associated with significantly lower prolactin levels during and throughout the infusion. There was a rebound to levels significantly higher than control on cessation of the infusion. Cortisol levels were unaffected by dopamine.

Diel and seasonal changes in resting plasma cortisol levels in juvenile Atlantic salmon, Salmo salar L.

Plasma cortisol levels in juvenile Atlantic salmon, Salmo salar L., were higher at night than during the day from September to May, and were higher in the morning than at night from June to August. Mean nocturnal levels increased slightly from August (less than 1.4 ng/ml) to January (18.2 ng/ml), sharply to a peak in March (46.0 ng/ml), and then declined sharply again reaching minimal values in July (less than 1.4 ng/ml). With the exception of a February peak (7.7 ng/ml) mean diurnal levels also increased slightly from a September minimum of (less than 1.4 ng/ml) to April (3.3 ng/ml), then more sharply to plateau from May to July (8.2-11.0 ng/ml), and declined again to September. The data support the hypothesis that rising cortisol levels in spring represent a generalized stress response to behavioral and physiological maladaptation at smolting.

Application of solid-phase antibodies to radioimmunoassay. Evaluation of two polymeric microparticles, Dynospheres and nylon, activated by carbonyldiimidazole or tresyl chloride.

Two types of polymeric microparticle, Dynospheres and reprecipitated acid-hydrolysed nylon 6/6, and two methods of activating these particles with either tresyl chloride or carbonyldiimidazole (CDI) prior to covalent linkage of antibodies were investigated with a view towards their respective adoption for the preparation of general solid-phase reagents for immunoassay applications. Activation of each particle and coupling of antibodies was rapid irrespective of the activator. CDI proved to be the activator of choice since it was cheap, less hazardous, more efficient and less pH dependent than tresyl chloride. Both types of microparticle remain buoyant during the RIA incubation periods and form stable pellets after centrifugation. In second antibody applications immobilisation of the first antibody occurs with a short incubation period of 30 min. Nylon microparticles have a higher antibody-coupling capacity and are the particles of choice in both first and second antibody applications. However, the nylon microparticles possess marginally higher non-specific binding characteristics.

Development and evaluation of a simple, direct, solid-phase radioimmunoassay of serum cortisol from readily available reagents.

A simple, rapid solid-phase radioimmunoassay for serum cortisol was developed using cortisol antibody distributed by the Scottish Antibody Production Unit and commercially available radioiodinated cortisol ligand. The assay involves a 1-h incubation at ambient temperature, using the antibody covalently linked by the easily performed carbonyldiimidazole method, to microcrystalline cellulose. A detailed comparison of the accepted 0.125 mol/l citrate, pH 4.0, and an alternative 0.1 mol/l phosphate/8-anilinonaphthalene sulphonic acid, pH 7.4, diluent demonstrated similar precision (within-assay 10% to 5%; between-assay 11% to 6% over the range 100-1000 nmol/l cortisol) and recovery (median recoveries: phosphate 98.6%, citrate 99.5%). Linear regression analysis of human serum cortisol concentrations in comparison with the 'Amerlex' cortisol RIA kit gave the following results: citrate = 1.029. 'Amerlex' - 3.5 nmol/l (n = 80, r = 0.967); phosphate = 1.017, 'Amerlex' + 26.2 nmol/l (n = 80, r = 0.961). Phosphate, pH 7.4 diluent was adopted as the diluent of choice, since it was economical of antibody and maintained good precision over a wider working range of cortisol concentration.