Ligand Immunofunctional Assay Research Articles

Evaluation of binding capacity of circulating insulin-like growth factor binding protein-1b in salmonids using a ligand immunofunctional assay

Insulin-like growth factor-binding proteins (IGFBPs) regulate the activity of insulin-like growth factor (IGF)-1. Among the three major circulating IGFBPs in salmonids, IGFBP-1b is an inhibitor of IGF activity induced under catabolic conditions. IGFBP-1b is considered to quickly sequester IGF-1 from the circulation. However, the level of circulating IGFBP-1b present in its unoccupied free form is unknown. Here, we aimed to develop a non-equilibrium ligand immunofunctional assay (LIFA) to evaluate IGF-binding capacity of circulating intact IGFBP-1b. Purified Chinook salmon IGFBP-1b, its antiserum, and europium-labeled salmon IGF-1 were used as the assay components. In the LIFA, IGFBP-1b was first captured by the antiserum, allowed to bind to the labeled IGF-1 for 22 h at 4 °C, and quantified its IGF-binding capacity. Serial dilutions of the standard and serum were prepared simultaneously within a certain concentration range (1.1–12.5 ng/ml). In underyearling masu salmon, IGF-binding capacity of intact IGFBP-1b was higher in fasted fish than in fed fish. Transferring Chinook salmon parr to seawater also increased IGF-binding capacity of IGFBP-1b, most likely due to osmotic stress. In addition, there was a strong relationship between total IGFBP-1b levels and its IGF-binding capacity. These results suggest that IGFBP-1b expressed under stress is mostly present in the free form. On the contrary, during smoltification of masu salmon, IGF-binding capacity of IGFBP-1b in the serum was relatively low and less related to the total IGFBP-1b level, suggesting its functional difference under certain physiological conditions. These results indicate that estimating both total IGFBP-1b level and its IGF-binding capacity is useful for evaluating the catabolic status and unraveling the regulation of IGF-1 activity by IGFBP-1b.

Intact rather than total circulating insulin-like growth factor binding protein-1a is a negative indicator of growth in masu salmon.

Insulin-like growth factor binding protein (IGFBP)-1a is one of three major circulating forms in salmon and induced under catabolic conditions. However, there is currently no immunoassay available for this form because of a lack of standard and specific antibodies. We developed a time-resolved fluoroimmunoassay (TR-FIA) for salmon IGFBP-1a using recombinant protein for labeling, an assay standard, and production of antiserum. The TR-FIA had a low cross-reactivity (3.6%) with IGFBP-1b, another major form in the circulation. Fasting for 4 wk had no effect on serum immunoreactive (total) IGFBP-1a levels in yearling masu salmon, whereas 6-wk fasting significantly increased it. There was a significant, but weak, negative relationship between serum total IGFBP-1a level and individual growth rate (r2 = 0.12, P = 0.01). We next developed a ligand immuno-functional assay (LIFA) using europium-labeled IGF-I to quantify intact IGFBP-1a. In contrast to total IGFBP-1a, serum intact IGFBP-1a levels increased after 4 wk of fasting, and refeeding for 2 wk restored it to levels similar to those of the fed control. Serum intact IGFBP-1a levels showed a significant negative correlation with individual growth rate (r2 = 0.52, P < 0.001), which was as good as that of IGFBP-1b. Our findings using newly developed TR-FIA and LIFA suggest that regulation of intact IGFBP-1a levels has an important effect on growth in salmon and that intact IGFBP-1a is a negative index of salmon growth.

High Circulating Leptin Receptors with Normal Leptin Sensitivity in Liver-specific Insulin Receptor Knock-out (LIRKO) Mice

Liver-specific insulin receptor knock-out (LIRKO) mice display hyperinsulinemia, abnormal glucose metabolism, and progressive liver dysfunction. In addition, circulating leptin levels appear to be increased more than 10-fold. However, food intake, body weight, and adipose mass are not significantly altered in LIRKO mice compared with wild-type littermates. Using a ligand immunofunctional assay, we found that the apparent increase in circulating leptin in LIRKO mice is because of an 80-fold increased serum level of soluble leptin receptor. Gene expression analysis by microarray and real time PCR reveals the liver as the source of soluble leptin receptor in LIRKO mice, with an increase in expression of the short (Ob-Ra), long (Ob-Rb), and soluble (Ob-Re) forms of the leptin receptor. Direct control of leptin receptor expression by insulin could also be demonstrated in isolated hepatocytes from normal mice. Despite the markedly increased levels of leptin receptor in their circulation, LIRKO mice exhibit normal or even enhanced leptin sensitivity, as assessed by their physiological and molecular responses to exogenous leptin administration and their lower base-line hypothalamic levels of SOCS3 mRNA. Thus, insulin signaling in the liver plays an important role in control of leptin receptor expression and shedding. In the LIRKO mouse, this is lost, leading to markedly increased leptin receptors into the circulation. These high levels of circulating leptin receptor bind leptin and likely alter its clearance, but do not inhibit leptin action and may actually potentiate leptin action. In this manner, insulin signaling in liver plays an important role in leptin homeostasis and fine modulation of leptin action.

Different isoforms of the soluble leptin receptor in non-pregnant and pregnant mice

Leptin circulates in murine serum in a free and a bound form. As shown in humans, a soluble leptin receptor (sOB-R), which modulates the effects of its ligand, circulates in murine blood. The aim of our study was to determine abundance and biochemical nature of this protein. For the quantification of sOB-R we developed a ligand-immunofunctional assay (LIFA) which is based on both, leptin binding and immunological recognition. The use of this LIFA revealed that during late gestation sera of pregnant mice had a ∼290-fold higher level of sOB-R than non-pregnant animals. As investigated by size exclusion chromatography these mice sera demonstrated a co-elution of their leptin binding activity with leptin immunoreactivity and levels of sOB-R measured by LIFA. Therefore, it has to be concluded that sOB-R represents the major leptin binding activity in murine circulation. The molecular analysis of sOB-R by Western blot and by cross-linking with 125I-leptin in sera of pregnant and non-pregnant mice demonstrated two different isoforms of sOB-R, which were capable of leptin binding. The sOB-R in serum migrated at a molecular weight of 150 kDa in pregnant and only of 120 kDa in non-pregnant animals. Deglycosylation of these isoforms led to sOB-R molecules which were found at the same molecular weight in SDS–PAGE. This finding indicates that both isoforms differ only in the degree of their glycosylation. In conclusion, the non-pregnant and the pregnant states are accompanied by differently glycosylated isoforms of sOB-R whose physiological relevance remains to be determined.

Growth hormone binding protein in normal and aneuploid pregnancy: a paradoxical decrease in trisomy 18

Objective To explore whether abnormalities in growth hormone binding protein (GHBP) may underlie the growth restriction associated with fetal aneuploidy. Design A retrospective casecontrol study. Setting Monash Medical Centre, Clayton, Victoria, Australia. Population Twenty-one trisomy 18, and 30 trisomy 21 pregnancies, and 170 chromosomally normal pregnancies at 15–18 weeks of gestation representing three to five controls per case matched for source, gestation and duration of storage. Methods GHBP was measured using a ligand immunofunctional assay Results In the chromosomally normal pregnancies GHBP levels decreased slightly but significantly across the narrow gestational window studied. Compared with controls, levels of GHBP, expressed as median (95% CI) multiples of the median (MoM), in the trisomy 21 pregnancies were similar, 1.0 (0.92–1.39) MoM and 1.27 (1.04–1.50) MoM, respectively; P=0.061 (Mann-Whitney U test) but were significantly reduced in the trisomy 18 pregnancies, 0.68 (0.51–0.84) MoM; P=0.0014 (Mann-Whitney U test). Conclusions These data suggest that decreased levels of maternal growth hormone binding protein, and by implication growth hormone receptor complement, may underlie the early severe growth restriction that is characteristic of trisomy 18.

Use of the ligand immunofunctional assay for human insulin-like growth factor ((IGF) binding protein-3 (IGFBP-3) to analyze IGFBP-3 proteolysis and igf-i bioavailability in healthy adults, GH-deficient and acromegalic patients, and diabetics.

The ligand immunofunctional assay for plasma insulin-like growth factor (IGF) binding protein (IGFBP)-3 developed in our laboratory provides for specific measurement of intact, as opposed to proteolyzed, IGFBP-3. IGFBP-bound IGFs are dissociated and separated by acid pH ultrafiltration; thereafter, intact and proteolyzed IGFBP-3 are captured by a monoclonal antibody in a solid-phase assay and incubated with (125)I-IGF-I, which detects the intact protein but not its proteolytic fragments. This assay was combined with assays for IGF-I (RIA of the ultrafiltrate) and total IGFBP-3 (immunoradiometric assay) to quantify the percentage of proteolyzed IGFBP-3 (percent proteolyzed IGFBP-3) and to calculate the IGF-I/intact IGFBP-3 ratio as an index of the fraction of exchangeable IGF-I bound to IGFBP-3. This fraction represents most of the IGF-I that is bioavailable. Because GH and insulin control the hepatic production and plasma concentrations of IGF-I and IGFBP-3, we set out to determine whether variations in the secretion of the two hormones are involved in the regulation of IGFBP-3 proteolysis. The study included adult populations of 36 healthy subjects, 23 hypopituitary patients untreated with GH, 43 acromegalics (13 untreated), 42 insulin-treated type 1 diabetics [insulin-dependent diabetes mellitus (IDDM)] patients, and 50 type 2 diabetics [non-IDDM (NIDDM)] patients, 22 of whom were insulin-treated and the remaining 28 treated with sulfonylurea and/or metformin). Unlike IGF-I and (to a lesser extent) total IGFBP-3 levels, which decline with age, percent proteolyzed IGFBP-3 seemed relatively stable. In healthy adults, the mean +/- SEM was 29.4 +/- 1.9 for subjects less than 45 yr old and was slightly (but not significantly) lower, 25.7 +/- 3, for those of more than 45 yr. There was no difference between male and female subjects. In GH-deficient patients, despite severely depressed IGF-I levels, percent proteolyzed IGFBP-3 and IGF-I/intact IGFBP-3 ratios were within the normal range. Among acromegalics, percent proteolyzed IGFBP-3 was elevated: 36.6 +/- 3.3 for patients of less than 45 yr, 33.3 +/- 3.2 for patients of more than 45 yr (P = 0.02 vs. healthy subjects). Consequently, the effects of excessive IGF-I synthesis are exacerbated by the enlarged exchangeable fraction of IGFBP-3-bound IGF-I. There was no significant difference in percent proteolyzed IGFBP-3 between GH-deficient patients before and after GH treatment or between treated and untreated acromegalics. In IDDM patients, the means for percent proteolyzed IGFBP-3 were higher than those in healthy adults: 36.7 +/- 3.7 (P = 0.03) and 31.3 +/- 3.3 for subjects of less than 45 and more than 45 yr, respectively. In NIDDM patients, all of whom were more than 45 yr old, the means were 35.2 +/- 2.5 (P = 0.02) for insulin-treated patients and 33 +/- 2.5 for the group treated orally. Among the diabetics, increased IGFBP-3 proteolysis resulted in an IGF-I/intact IGFBP-3 ratio that was normal for IDDM patients of less than 45 yr and above normal (P = 0.01) for the others. Percentage proteolyzed IGFBP-3 and the IGF-I/intact IGFBP-3 ratio were inversely related to body mass index in IDDM patients (r = -0.42, P = 0.008; and r = -0.31, P = 0.05, respectively) and to percentage glycosylated hemoglobin in all insulin-treated diabetics (r = -0.25, P = 0.05; and r = -0.33, P = 0.008, respectively). There was also an inverse relationship between IGF-I/intact IGFBP-3 ratios and IGFBP-1 levels in healthy adults (r = -0.39, P = 0.03) and orally treated NIDDM patients (r = -0.37, P = 0.05). Percentage proteolyzed IGFBP-3 was positively correlated to total IGFBP-3 in healthy adults (r = 0.65, P = 0.0001) and in all the groups of patients. It was negatively correlated to IGF-I/total IGFBP-3 in healthy subjects (r = -0.40, P = 0.02) and diabetics (r = -0.30, P = 0.005). This suggests an autoregulatory mechanism controlling the bioavailability of IGFBP-3-bound IGF-I in the 140-kDa complexes. In the pathological conditions studied here, regulation of IGF-I bioavailability by limited proteolysis of IGFBP-3 contributes toward an appropriate adaptation to insulin deficiency and/or resistance but not to disturbances of GH secretion.

Soluble Leptin Receptor Represents the Main Leptin Binding Activity in Human Blood

In human blood leptin circulates both free and bound to high molecular weight proteins. Hypothesising that these proteins may modulate ligand bioavailability and bioactivity of leptin, we investigated their molecular nature. Therefore, leptin binding activity was partially purified from human plasma using a leptin affinity column. Subjecting this preparation to size exclusion chromatography (SEC) we observed a coelution of leptin binding activity with levels of the soluble leptin receptor (sOB-R) determined by a newly developed ligand immunofunctional assay. In Western blot analysis the partially purified leptin binding activity exhibited sOB-R immunoreactivity in two bands of 110 and 140 kD. Following N-deglycosylation these bands were replaced by two bands with the molecular weight of 90 and 60 kD, suggesting two isoforms which are capable of leptin binding, as determined by cross-linking. Furthermore, different ratios of these isoforms were detectable in fractions of the leptin binding activity after separation by SEC. These findings indicate the formation of heterodimers and homodimers complexed with and without leptin. As the two sOB-R bands from Western blot analysis correspond to only two specific bands in cross-linking experiments with 125l-leptin, the role of both isoforms as leptin binding proteins appears to be exclusive. Therefore, our results indicate that sOB-R is the major leptin binding protein in the circulating human blood.

Measurement of intact insulin-like growth factor-binding protein-3 in human plasma using a ligand immunofunctional assay.

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is a fundamental mechanism in the regulation of IGF-I bioavailability in the bloodstream. Its measurement by Western immunoblotting provides only semiquantitative estimation. We have developed a ligand immunofunctional assay (LIFA) for quantifying human (h) intact IGFBP-3 in biological fluids. IGFBP-bound IGFs are dissociated and separated by acid pH ultrafiltration, and a monoclonal antibody specific to the first 160 amino acids of IGFBP-3 is used to capture hIGFBP-3 in a solid-phase assay. The complex is then incubated with (125)I-IGF-I, which binds to intact IGFBP-3 but not to its proteolytic fragments. Binding specificity was demonstrated in competition experiments with unlabeled IGF. Nonspecific binding was 1.4%. The fragments comprising residues 1-160 and 1-95 of recombinant hIGFBP-3 [corresponding to the major proteolytic fragments of approximately 30 kDa and (glycosylated) 20 or (nonglycosylated) 16 kDa detected in serum by Western immunoblotting, respectively] fail to bind (125)I-IGF-I when complexed with the monoclonal antibody. Similarly, no binding of (125)I-IGF-I was obtained in the LIFA when applied to plasmas from pregnant women during the final 3 months of pregnancy, where the characteristic 42- to 39-kDa doublet of intact IGFBP-3 is undetectable. The standard curve was established using a pool of plasmas (EDTA) from healthy adults, for which standardization with glycosylated recombinant hIGFBP-3 yielded an intact IGFBP-3 content of 2 microg/mL. The dynamic range of the LIFA was 0.50-3.75 microL equivalent of the plasma pool in a total volume of 300 microL per assay tube, with a sensitivity threshold of approximately 1 ng intact IGFBP-3. Unknown plasma samples were studied at three concentrations. Intra- and interassay variations were 3.6% and 4%, respectively. In 31 healthy adults, the mean plasma concentration of intact IGFBP-3 was 2.24 +/- 0.08 (SEM) mg/L, and that of total IGFBP-3 measured by immunoradiometric assay was 3.27 +/- 0.14 mg/L. The calculated mean proportion of proteolysed IGFBP-3 was 29.4 +/- 1.9%. In these subjects, a close correlation was found between intact and total IGFBP-3 (r = 0.71, P = 0.0001). The LIFA for IGFBP-3, therefore, provides accurate and sensitive measurement of intact IGFBP-3, the form with the functional capacity to sequester IGF-I in the bloodstream by association with the acid-labile subunit in 140-kDa complexes. In combination with total IGFBP-3 and IGF-I assays, the LIFA opens new perspectives in investigating the regulation of IGFBP-3 proteolysis and IGF-I bioavailability in man.

Growth hormone-binding protein in normal and pathologic gestation: correlations with maternal diabetes and fetal growth.

To date, measurements of GH-binding protein (GHBP) during human pregnancy have been carried out using assays susceptible to interference by the elevated levels of human placental GH typical of late gestation. We recruited a large cohort of pregnant women (n = 140) for serial measurements of GHBP and used the ligand immunofunctional assay for GHBP. For normal gravidas, GHBP levels fell throughout gestation. Mean levels were 1.07 nmol/L (SE = 0.18) in the first trimester, 0.90 nmol/L (SE = 0.08) at 18-20 weeks, 0.73 nmol/L (SE = 0.05) at 28-30 weeks, and 0.62 nmol/L (SE = 0.06) at 36-38 weeks. GHBP levels in the first trimester correlated significantly with maternal body mass index (r = 0.58; P < 0.01). GHBP levels in pregnancies complicated by noninsulin-dependent diabetes mellitus (NIDDM) were substantially elevated at all gestational ages. The mean value in the first quarter (2.29 nmol/L) was more than double the normal mean (P < 0.01). In contrast, patients with insulin-dependent diabetes mellitus (IDDM) showed reduced GHBP concentrations at 36-38 weeks. The correlation between body mass index and GHBP is consistent with a metabolic role for GHBP during pregnancy, as is the dramatic elevation in GHBP observed in cases of NIDDM. At 36 weeks gestation, GHBP was significantly elevated (P < 0.01) in those women whose neonates had low birth weight (< 10th percentile). In early gestation (< 14 weeks), GHBP tended to be higher in women whose fetuses were designated to be at risk of intrauterine growth retardation (1.39 nmol/L; n = 4; compared with 1.07 nmol/L in normals), but this did not reach statistical significance. Although both NIDDM and IDDM pregnancies are at risk of fetal macrosomia, their GHBP concentrations are markedly divergent. This paradox and the roles of glucose and insulin in the regulation of GHBP during gestation warrant further investigation.

Measurement of growth hormone-binding protein in the rat by a ligand immunofunctional assay

Measurement of growth hormone-binding protein in the rat by a ligand immunofunctional assay

Control of growth hormone (GH) binding protein release from human hepatoma cells expressing full-length GH receptor.

In humans and rabbits, the circulating GH binding protein (GHBP) is released from the GH receptor by cleavage at a site proximal to the cell surface. There is evidence that GHBP status is predictive of GH responsiveness, presumably because it reflects GH receptor status. This assumes that GHBP release is not a regulated step. Here we report a model for study of GHBP release that provides some insight into this question. Human HepG2 cells were stably transfected with rabbit GH receptor and shown to be responsive to nonprimate (bovine) GH, indicating functionality of the transfected receptor. These cells released GHBP of the expected size, and this release could be increased by incubation with a phorbol ester, which stimulated receptor synthesis through the cytomegalovirus promoter. We surveyed a wide range of protease inhibitors both with and without streptolysin-O permeabilization, with the intention of defining the endogenous protease. Of 16 inhibitors, only benzamidine proved an effective inhibitor of release, indicating the existence of a novel protease. We could increase GHBP release with a membrane impermeable thiol blocker, suggesting activation of a membrane protease. We examined the ability of IGF-1, insulin, dexamethasone, sex steroids, and T4 to influence GHBP release. Although these agents are known to be effective in the parent hepatoma line, none were effective in modulating GHBP release, although GH itself decreased release by around 30% as assessed with a ligand immunofunctional assay. We conclude that GHBP release appears to be constitutive in this model and driven by receptor availability. This is consistent with an in vivo situation where circulating GHBP provides an index of hepatic receptor expression.

Measurement of growth hormone-binding protein in the rat by a ligand immunofunctional assay.

We have developed a ligand immunofunctional assay (LIFA) for quantifying the circulating functional GH-binding protein (GHBP) in the rat. This two-site solid-phase assay uses a capture monoclonal antibody (4.3) specific to the hydrophilic C-terminal segment of rat GHBP (rGHBP), saturation of binding with human GH, and a detection system of rabbit antihuman GH polyclonal antibody and peroxidase-conjugated antirabbit immunoglobulin G antibody. Results were compared with Scatchard estimates derived by immuno-precipitation with monoclonal antibody 4.3. This assay was used to determine the GHBP levels in male and female rats and to investigate the diurnal properties and dynamics of GH and GHBP interaction in 15-min blood sampling over a 6-h period. The dynamic range of the rLIFA was 0.15-20.0 nM recombinant rGHBP, with intraassay and interassay coefficients of variation of 10.5% (n = 20) and 12.9% (n = 12), respectively. Serum GHBP levels determined by the rLIFA and those derived from Scatchard estimates were strongly correlated (n = 8; beta = 0.55; r2 = 0.89; P = 0.0005). Male rats had lower GHBP levels (6.5 +/- 0.7 nM; mean +/- SE; n = 14) than female rats (35.4 +/- 2.7 nM; n = 15; P = 0.0001). In the diurnal study, male rats had higher GH peaks (312.5 +/- 121.6 ng/ml; n = 7) than female rats (96.5 +/- 15.4 ng/ml; n = 9; P < 0.0001). In contrast to the pulsatile secretion of GH, GHBP levels in both sexes remained stable and showed no relationship to secretory pulses of GH. However, the GH bursts significantly altered the distribution of the GH-GHBP complex in male rats. By saturation and mass analysis, the greater GH pulsatile secretion in male rats resulted in occupancy of GHBP from less than 5% at nadir to about 80% at secretory peaks, in contrast to the less than 5-15% range of GHBP occupancy in female rats. In male rats, greater than 80% of GH at secretory peaks existed in the free form, whereas in female rats, 16-23% of GH existed in the free form during pulsatile secretion. In summary, the rLIFA shows good correlation to Scatchard analysis using an identical antibody. We conclude that this assay provides a rapid, sensitive, and accurate measurement of the circulating functional GHBP in the rat, and that it facilitates the study of GH and GHBP dynamics under a range of physiological conditions.

Direct quantitation of growth hormone binding protein in human serum by a ligand immunofunctional assay: comparison with immunoprecipitation and chromatographic methods.

Current methods for measuring GH-binding protein (GHBP) are laborious, require separation of GHBP complex, and may be affected by endogenous GH content of the sample. Such methods estimate binding activity and GHBP concentration can only be obtained by Scatchard analysis. We have developed and validated a 2-site immunofunctional assay for the direct quantitation of GHBP in human serum employing a capture monoclonal antibody against GHBP and a polyclonal antibody against hGH. Results were compared with binding activity data and Scatchard-derived capacity estimates obtained by immunoprecipitation and gel chromatography procedures. Serum samples were obtained from 21 subjects with GH deficiency, 24 patients with acromegaly, and 56 normal subjects; 12 of whom were postmenopausal women who were studied before and during oral estrogen treatment. Using the immunofunctional assay, serum GHBP concentrations in normal subjects ranged from 0.14-3.28 nmol/L, was positively related to body mass index (BMI, P = 0.0004) and negatively related to age (P = 0.015). Women had significantly higher levels (0.99 +/- 0.12 vs. 0.63 +/- 0.09 nmol/L; P = 0.0191) than age and BMI-matched men. GHBP levels were not different between normal, acromegalic, or GH-deficient subjects. Oral estrogen therapy in postmenopausal women increased serum GHBP concentrations up to 5-fold. There was a significant nonlinear relationship between the immunofunctional assay measurements and binding activity by either immunoprecipitation (r = 0.84) or chromatographic (r = 0.73) methods; increase in GHBP concentrations was not reflected in proportionate increase in both activity assays. Estrogen-induced changes in circulating GHBP levels were greatest with the immunofunctional assay followed by Scatchard-derived values from immunoprecipitation and chromatographic methods. We conclude that ligand immunofunctional assay measurements of GHBP are higher but show good agreement with binding activity or Scatchard derived estimates from immunoprecipitation and chromatographic methods. This assay provides a direct and practical tool for rapid, accurate and sensitive estimation of GHBP concentration in serum.

Direct quantitation of growth hormone binding protein in human serum by a ligand immunofunctional assay: comparison with immunoprecipitation and chromatographic methods

Direct quantitation of growth hormone binding protein in human serum by a ligand immunofunctional assay: comparison with immunoprecipitation and chromatographic methods