Serum leptin concentration during puberty in healthy nonobese adolescents

Data obtained during the past five years have indicated that there are important age- and gender-based differences in the regulation and action of leptin in humans. To study the physiological changes of leptin during puberty in both sexes, and its relationship with body composition and sexual maturation, we measured leptin concentrations in 175 healthy adolescents (80 girls, 95 boys, 10-18 years of age), representing all pubertal stages. We excluded individuals with a body mass index (BMI) below the 5thor above the 95th percentile relative to age. Serum concentrations of leptin were determined by a monoclonal antibody-based immunofluorimetric assay, developed in our laboratory. Body composition was determined by dual-energy X-ray absorptiometry. Pubertal stage was assigned by physical examination, according to Tanner criteria for breast development in females and genital development in males. Leptin concentration in girls (N = 80) presented a positive linear correlation with age (r = 0.35, P = 0.0012), BMI (r = 0.65, P < 0.0001) and percentfat mass (r = 0.76, P < 0.0001). In boys (N = 95) there was a positive correlation with BMI (r = 0.49, P < 0.0001) and percentfat mass (r = 0.85, P < 0.0001), but a significant negative linear correlation with Tanner stage (r = -0.45, P < 0.0001) and age (r = -0.40, P < 0.0001). The regression equation revealed that percentfat mass and BMI are the best parameters to be used to estimate leptin levels in both sexes. Thus, the normal reference ranges for circulating leptin during adolescence should be constructed according to BMI or percentfat mass to assure a correct evaluation

A specific and highly sensitive time-resolved fluoroimmunoassay for human proinsulin

We describe a time-resolved fluoroimmunoassay specific for human proinsulin using a combination of two high-affinity monoclonal antibodies, one against insulin and the other specific for intact proinsulin and for split 65-66 and des 64-65 proinsulin forms. The assay employs only 200 micro liters of serum, with a detection limit of 0.1 pmol/l. The intra-assay variation coefficient was less than 3 percent between 3 and 1000 pmol/l. There was 0 percent cross-reaction with insulin, C-peptide, split 32-33 and des 31-32 proinsulin. Serum concentration of proinsulin was analyzed in 50 subjects during an oral glucose tolerance test (10 non-obese controls, 10 obese controls, 10 subjects with impaired glucose tolerance, 10 patients with type II diabetes meIlitus (DM) and fasting blood glucose (FBG) <140 mg/dl, and 10 patients with type II DM and FBG >150 mg/dl). Mean fasting serum proinsulin levels measured by this assay in non-obese controls (0.84 +/-0.90 pmol/l; 0.1-2.4 pmol/l) were lower than the results reported by her investigators. There was an increase of proinsulin related to obesity and increased glucose levels, suggesting that proinsulin levels increase with insulin resistance.

Monoclonal antibodies specific for the free alpha subunit of glycoprotein hormones and their use in the development of a sensitive immunofluorometric assay

Glicoprotein hormone free alpha subunit has been used as a marker for some pituitary tumors and to study the reactivity of glycoprotein hormone-producing cells under different circunstances. We describe a highly sensitive ans specific immunofluorometric assau for the measurement of serum free alpha subunit levels. The assay is based on a monoclonal antibody, specific for free alpha subunit, bound to microtiter plates. As tracer antibody we employed an europium-labelled free/complexed alpha subunit specific monoclonal antibody. Using overnight incubation and 50µl samples, the least detectable dose was of the order of 4 ng/1. Cross-reactivity with LH, TSH, FSH, and hCG was 6.5, 1.2, 4.3 and 1.1 percent, repectively. Normal adult males showed values ranging from 120 to 790ng/l, not different from normal adult premenopausal females (88 to 604 ng/l). In post-menopausal females, serum concentrations were significantly highler, ranging from 341 to 407 ng/l. In 56 patients with untreated pituitary tumors (18 "non-secreting", 25 GH-producing and 13 prolactin-producing tumors), 10 showed high values, 3 of them from the first group, 3 from the second and 4 from the third. We conclude that this highly sensitive assay can be a valualbe tool for the diagnosis and follow-up of selected patients with pituatary tumors and in other circumstances in which the glycoprotein hormone-producing cells of the pituitary require evaluation

Comparison of the determination of insulin by a monoclonal antibody-based immunofluorometric assay and by radioimmunoassay

This paper describes an immunofluorometric assay (IFMA) for insulin and compares it with the classical radioimmunoassay (RIA). Monoclonal antibodies against insulin were produced and used to develop the IFMA. One, immobilized on microtiter plates, was used for capture, the other, labelled with Europium, was used as tracer antibody. The IFMA presentes sensitivity to an amount of insulin of 3 pmol/1 and acceptable valueus for intra- and interassay error. The IFMA presented superimposable curves for human insulin, Arg65/Gly66-split proinsulin and des-Lys64, Arg65, and no cross-reactivity with human proinsulin, Arg32/Glu33 -split and des-Arg31, Arg32. The RIA showed 100 percent cross-reactivity with human proinsulin, 90 pecent with des-Arg31, Arg32 and 170 percent with des-Lys64, Arg65. The assay were used to measure insulin in 300 serum samples from 50 subjects submitted to an oral glucose tolerance test (OGTT). Twenty were normal, 10 had impaired glucose tolerance and 20 non-insulin-dependent diabetes mellitus. The mean value (ñ SEM) obtained bu IFMA was 166.7 ñ 12.1 pmol/1 and the mean value obtained by RIA was 339.6 ñ 18.6, with a correlacion of r = 0.80 (P0.01). Comparison of basal insulin levels of the different groups of individuals using IFMA or RIA led to the same conclusions. The area under curve showed statistically significant differences only for the comparison between normal lean subjects and individuals with impaired glucose tolerance, when measured by RIA...(au)

Monoclonal antibody-based immunoenzymometric assay for serum human growth hormone

A monolconal antibody-based immunoenzymometric assay (IEMA) for the measurement of human serum growth hormone is described. Two high-affinity and complementary monoclonal antibodies were selected from a panel of 9 obtained upon fusion of SP/O myeloma cells with spleen cells from a Balb/c mouse immunized against human growth hormone of pituitary origin. One monoclonal antibody was immobilized by attaching it to the walls of microtiter wells and the second was biotinylated. The reaction was quantitated by the addition of streptavidin-peroxidase. The sensitivity of the assay was 0.2 mIU/1 and the intra-and interassay coefficients of variation for 4.6 to 46 mIU/I wee < 8.3 and 17.3%, respectively. Cross-reaction with human placental lactogen, human prolactin and rat growth hormone was < 0.1% (w/w). Comparison of results obtained for 180 routine serum assays by radioimmunoassay and the assay described here had a correlation coefficient of 0.94 with a mean value of 16.3 ñ 1.3 (X ñ SEM) and 13.3 ñ 1.2 mIU/!, with the IEMA providing values 18% lower than the RIA. The discrepancy emphasizes the necessity of redefining normal ranges before immunometric assays, like the one described, can be used routinely