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Objective: Determination of insulin-like growth factor (IGF)-I is now a routine adjunct to multiple research and clinical investigations. Evidence has associated higher IGF-I levels with various human pathologies, but the reported associations have not been invariably confirmed. We examined the potential for post-sampling proteolysis and evaluated the impact of such events on IGF-I immunoassays. Design and methods: We compared IGF-I in different sets of fresh and frozen old samples using four different and commonly used immunoassays. The potential for post-sampling proteolysis was further examined by assaying fresh samples stored for 4 weeks at various temperatures in the absence or presence of protease inhibitors. Results: IGF-I levels in fresh serum samples from adult males, females, and pregnant subjects by all methods were similar and were highly correlated (r = 0.85–0.97). The same was true for levels in frozen (∼2 years at −80°C) samples from diabetic patients, which are reportedly associated with enhanced proteolytic activity. In contrast, in another set of frozen adult male and female samples (∼8 years at −20°C), the inter-method median IGF-I levels varied by ∼3- to 4-fold and the values poorly correlated. Similar variability in the inter-method response was also observed when IGF-I in the replicates of fresh samples stored at 4°C for 4 weeks was measured. However, the 4°C storage effect could be completely blocked by the addition of protease inhibitors, allowing for all assays to detect 92–101% of the expected mean levels. Conclusions: The data indicate susceptibility of IGF-I to significant post-sampling proteolysis and suggest the importance of immunoassays for the intact molecule. Immunoassays that lack specificity for intact IGF-I may mask the potential pathophysiological effects of proteolysis and generate misleading results, particularly in studies involving inappropriately stored and/or proteolyzed samples. In such cases, underestimation of the in vivo levels by the intact assays would occur, but the findings of low IGF-I levels may be indicative of questionable sample quality.

Objective: Inhibin circulates in various molecular weight forms. Alpha (α)-subunit-directed total inhibin immunoassays, which detect all forms of α subunits plus the α/β inhibin dimers, have been found valuable in the diagnosis and monitoring of ovarian cancer. Because of the dependency of the published methods on boiling sample pre-treatment with SDS and unavailability of a commercial assay, we developed an enzyme-linked immunosorbent assay (ELISA) for direct determination of total inhibin. Design and methods: Method development involved a pair of well-characterized inhibin α subunit-directed antibodies and determination of the effects of various assay parameters. Selection of the optimized protocol was guided by the outcome of comparative sample analysis using previously reported boiling sample pre-treatment reagents and protocols. Results: We report development of a simplified ELISA for total inhibin. Method evaluation data demonstrated acceptable analytical performance characteristics with detection limit of 2 ng/l (recombinant inhibin-A), dynamic range of 12.5–500 ng/l, and intra- and inter-assay imprecision of 2.3–4.6% and 3.3–5.1% at total inhibin concentrations of approximately 60–400 ng/l, respectively. The mean (±SD) recovery from spiked serum samples averaged 109 ± 14% and recovery in response to serial sample dilution was 99 ± 10%. Serum values by the direct method ( n = 40) correlated strongly with those obtained after sample pre-treatment by boiling with SDS ( r = 0.97). As expected, the total inhibin immunoreactivity in human follicular fluid fractionated by HPLC gel filtration in multiple immunoreactive peaks (8–250 kDa). In serum samples from postmenopausal women with ovarian cancer, the assay detected significantly higher total inhibin levels than in samples from normal postmenopausal controls. Conclusion: The development of a fast and simplified ELISA should facilitate wider investigations of pathophysiology and diagnostic potential of total inhibin measurement.

The IGF-binding protein (IGFBP)-related proteins (rPs) are a group of recently described cysteine-rich proteins that share significant amino-terminal structural similarity with the conventional IGFBPs. IGFBP-rP1 (also known as MAC25/angiomodulin/prostacyclin-stimulating factor and T1A12), regulates cellular proliferation, adhesion, and angiogenesis and stimulates prostacyclin synthesis. We characterized new monoclonal antibodies generated against IGFBP-rP1 and have used them to study the distribution of IGFBP-rP1 in human biological fluids and tissues. Additionally, we have developed a noncompetitive sandwich-type immunoassay to quantitate the concentrations of IGFBP-rP1 in human serum. IGFBP-rP1 was readily detectable in serum, urine, amniotic fluid, and cerebrospinal fluid by immunoblot analysis. Evaluation of the newly developed immunoassay demonstrated acceptable analytical performance, with a detection limit of 0.7 micro g/liter, a dynamic range of 3.1-100 micro g/liter, and intra- and interassay coefficients of variation of 2.5-6.8% and 3.1-6.4% at approximately 24-85 ng/ml IGFBP-rP-1, respectively. No significant cross-reactivity with IGFBP-1-6 was observed. In random normal human adult sera (n = 37), the median IGFBP-rP1 was 21.0 micro g/liter, and values did not correlate with levels of IGF-I (r = 0.085, P = 0.61), IGF-II (r = 0.051, P = 0.75), or IGFBP-3 (r = 0.061, P = 0.74). The monoclonal anti-IGFBP-rP1 antibodies also readily detected IGFBP-rP1 expression in human tissue sections, with preferential expression of IGFBP-rP1 in the microvascular endothelium associated with tumorigenesis. In summary, using newly developed IGFBP-rP1 monoclonal antibodies, we confirm the presence of IGFBP-rP1 in the major human body fluids, provide quantitative normative data on the concentrations of IGFBP-rP1 in human serum, and show preferential expression of IGFBP-rP1 in the microvascular endothelium associated with tumorigenesis. The use of these novel IGFBP-rP1 detection tools should prove useful in the elucidation of the biological role(s) of this protein.

Markers of myocardial injury have been vital in the assessment of patients with coronary heart disease. Pregnancy associated plasma protein A (PAPP)-A is an insulin-like growth factor (IGF) binding protein (IGFBP)-4 protease and a potential early indicator of unstable angina. We developed an ultrasensitive enzyme-linked immunosorbent assay (ELISA) for PAPP-A and measured serum PAPP-A in patients with biochemical evidence of acute coronary syndrome.Method development was based on pair-wise evaluation of a panel of antibodies and determination of PAPP-A specificity and sensitivity relative to those of a conventional method. Association of PAPP-A with myocardial damage was assessed in serum samples classified based on serum creatine kinase (CK)-MB or cardiac troponin-T levels.Serum PAPP-A was significantly higher in samples with elevated CK-MB or troponin-T than in samples with normal CK-MB (p0.001). Marker-association studies showed strong correlation between PAPP-A and troponin-T (r = 0.59, p0.001) in a subset of troponin-T positive samples. Indications for both parallel as well as divergence in the expression of PAPP-A and troponin-T was also evident when serial timed samples available from a number of patients were analyzed.The data are consistent with the conclusion that expression of PAPP-A is enhanced in patients with biochemical evidence of acute coronary syndrome and suggest strongly that demonstration of PAPP-A association with other cardiac markers might be influenced by their relative release dynamics (timing and duration). The availability of the ultrasensitive PAPP-A ELISA should facilitate systematic investigations of PAPP-A expression in this and other pathophysiological conditions that might involve altered expression of the IGF/PAPP-A system.

Measurements of insulin-like growth factor I(IGF-I), IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) are important in assessing the GH-IGF axis. As nearly all IGF-I, IGFBP-3, and ALS circulate in a GH-dependent ternary protein complex, direct determination of the complex may be of significant analytical and clinical importance. We evaluated a panel of monoclonal antibodies (mAb) to human IGFBP-3 and classified them into four groups (G-1 to G-4). G-1 antibodies recognized epitopes that mapped at or near IGFBP-3 ligand (IGF)-binding site. This region overlapped with the G-2 defined region, which, in turn, overlapped with G-3 epitopes defined by one antibody (mAb 3). Only G-1 and G-3 antibodies paired without interference. mAb 9 recognized a conformational epitope (G-4), and mAb 10 was nonreactive. In pairwise mixed antibody evaluation, mAbs in G-2 and G-3 showed simultaneous binding to serum IGFBP-3 complexes in combination with an anti-IGF-I or an anti-ALS antibody. On this basis, two novel enzyme-linked immunosorbent assays (ELISAs) involving IGFBP-3/IGF-I (ELISA-1) and IGFBP-3/ALS (ELISA-2) recognition partners were developed, both demonstrating acceptable analytical performance characteristics. IGFBP-3 complexes measured by ELISA-1 and -2 in samples from normal individuals and subjects with GH deficiency, acromegaly, and GH receptor deficiency more tightly correlated with IGF-I, IGFBP-3, and ALS than IGF-II. ELISA-1 determinations were comparatively more age dependent and, in comparison to ELISA-2, showed better discriminations among the various sample groups, particularly among GH receptor deficiency, normal, and GH deficiency subjects. The development of IGFBP-3 complex ELISAs may simplify diagnostic applications and facilitate investigations of the physiological relevance of the ternary complex formation.

Development of an ELISA for phosphorylated isoform of IGFBP-1. Serine phosphorylation is an important regulator of IGFBP-1 bioactivity, but specific immunoassays for its measurement are currently lacking.Assay design was based on a novel approach of first capturing the phosphorylated and non-phosphorylated IGFBP-1 by an anti-IGFBP-1 antibody and then selectively detecting the phosphorylated form by an anti-phosphoserine antibody. Method development involved pair-wise evaluation of the candidate antibodies and determinations of analytical performance and specificity. Specificity was monitored by reactivity with dephosphorylated IGFBP-1, with antibodies against other phosphorylated residues that are not expressed, and by comparative analysis of sample containing different IGFBP-1 phosphorylation profile.Analytical evaluation demonstrated acceptable performance; detection limit 0.3 microg/L, dynamic range 1.56-100 microg/L; intra- and inter-assay CVs 2.1-8.6%; mean recovery (+/-SD) 97.8+/-9.2%, and mean recovery of sample dilution 93.4+/-6.0%. The phosphorylated and total IGFBP-1 medians in non-pregnant adult serum, which mostly contain the highly phosphorylated isoform, were 11.9 and 18.6 microg/L, respectively, and the sample values were tightly correlated (r=0.99). As expected, the corresponding medians in 1st trimester (17.4 and 63.0 microg/L) and 2nd trimester (30.9 and 75.8) samples with altered IGFBP-1 phosphorylation were significantly different (p0.001). Similarly, a fraction (1.29%) of total IGFBP-1 (13.3 mg/L) in amniotic fluids was found to be phosphorylated (0.172 mg/L). There was no reactivity with dephosphorylated IGFBP-1.The present ELISA is highly specific for the phosphorylated isoform of IGFBP-1 and its availability should help expedite further investigations of IGFBP-1 phosphorylation.

Objective: To examine the hypothesis that 12 weeks of resistance training would alter circulating concentrations of IGF-I system components in end-stage renal disease (ESRD) patients. Design: Ten ESRD patients underwent 12 weeks of resistance training after a 6 week control period and had morning fasted blood drawn on four occasions (weeks – 6, 0, 6, 12). Immunoassays were performed for serum total and free IGF-I, IGF binding proteins (IGFBPs) 2 and 3, and the acid labile subunit (ALS). Immunoaffinity depletion of ALS-based complexes allowed measurement of non-ternary (i.e., binary) IGF-I and IGFBP-3. Results: Significant improvements in strength and functional performance were observed. All IGF-I measures were stable during the control period and no changes were observed for the first 6 weeks of resistance training. At week 12, total IGF-I (−15.4 ± 28.9%), ternary IGF-I (−16.4 ± 36.7%), and the IGF-I/IGFBP-3 ratio had significantly (P⩽0.05) declined from week 0 values. No changes were observed for free IGF-I, IGFBPs 2 and 3, or the acid labile subunit. The proportion of IGF-I in ternary (∼76.3 ± 6.8%), non-ternary (∼22.5 ± 6.6%), and free (∼1.2 ± 0.5%) forms remained constant throughout the training. Conclusions: 12 weeks of resistance training in ESRD patients induced a decline in total IGF-I, but did not alter the proportion of IGF-I circulating in free, ternary or non-ternary molecular complexes. The decline in IGF-I occurs in the presence of positive training adaptations on physical performance and we conclude that this response pattern appears to be reflective of favorable neuromuscular anabolic adaptations.

Posttranslational modifications, particularly proteolysis, may play a significant role in the regulation of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) physiology, and thus, measurement of modified variants of IGFBP-3 and/or their combination ratios may have important research and diagnostic relevance. Based on evaluation of a panel of monoclonal and polyclonal IGFBP-3 antibodies, we constructed three new enzyme-linked immunosorbent assays (ELISAs) using a common capture and polyclonal (ELISA-3) or monoclonal (ELISA-1 and -2) detection antibodies and evaluated them in a two-step colorimetric procedure. Evaluation of ELISA-1-3 demonstrated detection limit, dynamic range, overall precision, and recovery of the added IGFBP-3 to be generally less than 0.04 microg/L, 2-100 microg/L, less than 10%, and 91-113%, respectively. IGF-I and -II, and IGFBP-1, -2, -4, -5, and -6 did not interfere. In normal adult sera (n = 26), seminal plasma (n = 14), pregnancy sera (n = 30), and amniotic fluid (n = 30), ELISA-1-3 detected significantly different IGFBP-3 levels (by up to 6-fold, on the average), whereas levels in seminal plasma determined by ELISA-1 were undetectable. Comparison of the values obtained vs. corresponding levels by an established method (Diagnostic Systems Laboratories, Inc., active IGFBP-3 ELISA) were similarly sample dependent and, on the average, varied by up to 19-fold. Only ELISA-3 compared well with the Diagnostic Systems Laboratories, Inc., IGFBP-3 ELISA when samples from normal adults were analyzed. The observed variability could not be totally explained by 50% lower reactivity of ELISA-1-3 for glycosylated IGFBP-3 vs. the nonglycosylated form, and changes in phosphorylation had no effect on immunoreactivity. Evaluation of IGFBP-3 after proteolysis by seminal plasma, plasmin, or thrombin suggested recognition of intact IGFBP-3 by ELISA-1, whereas ELISA-3 appeared to measure intact and proteolyzed IGFBP-3 (total IGFBP-3) with similar potency. In contrast, levels determined by ELISA-2 increased severalfold, indicating preferential recognition of IGFBP-3 fragments. We propose that immunoassay capable of differential determination of IGFBP-3 variants may help better define the physiological importance and potential clinical value of IGFBP-3 measurements.

To facilitate broader applications of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) analysis, we developed procedures for their measurements in extracts of whole blood dried on filter paper. A single 8-mm diameter filter paper disc containing about 13 microL blood was used. IGFBP-3 was efficiently extracted in a buffer within 1 h of incubation. IGF-I extraction involved incubation in buffer followed by acidification and neutralization steps. Blood spot assays showed intra- and interassay coefficients of variation (including interspot variations) of 5.4-16.7% for IGF-I and 6.6-11.7% for IGFBP-3; recoveries were 97 +/- 7.1% and 101 +/- 8.7%, respectively. Recoveries of IGF-I and IGFBP-3 in response to 4- to 8-fold variations in extraction buffer volume were 97 +/- 8.2% and 107 +/- 6.1%, respectively. Dried blood spot IGF-I and IGFBP-3 showed greater than 1-month stability at -20 C, 4 C, and room temperature and retained more than 65% of the immunoreactivity after approximately 1 month at 37 C. Both IGF-I and IGFBP-3 were contained within the plasma fraction of whole blood, and variations (mean +/- SD) in IGF-I (204 +/- 29 micrograms/L) and IGFBP-3 (4.4 +/- 0.48 mg/L) measured in extracts of dried blood spot with adjusted hematocrit of 0.2-0.62 were acceptable. IGF-I and IGFBP-3 in paired plasma and dried blood spot extracts of random samples (n = 46) showed excellent correlation (r0.94) with slopes of near unity. Compared to conventional methods, the filter paper procedures were equally effective in distinguishing IGF-I and IGFBP-3 levels in untreated GH receptor-deficient (n = 11) and age-matched normal controls (n = 16). We conclude that blood collected on filter paper is ideal for IGF-I and IGFBP-3 analysis and may find applications in pediatric and large scale infant screening programs.

Although the acid-labile subunit (ALS) of the approximately 150-kDa insulin-like growth factor (IGF)-binding protein (IGFBP) complex was described over a decade ago, details of ALS physiology have remained largely uncertain. We evaluated antibodies to synthetic human ALS and constructed a noncompetitive ALS enzyme-linked immunosorbent assay. Whereas uncomplexed ALS is directly measured, determination of total levels required sample pretreatment with SDS, which was found to optimally dissociate complexed ALS and significantly enhance ALS immunoreactivity. ALS in random adult sera was approximately 50% uncomplexed, and samples devoid of complexed ALS by immunoaffinity separation contained about 54% of the total levels. Serum ALS fractionated by gel filtration high performance liquid chromatography eluted in a single peak at approximately 150 kDa with IGF-I and IGFBP-3, but appeared at about 400-500 kDa after sample acidification and fractionation under acidic condition. The unexpected shift in ALS immunoreactivity remained unchanged when acid-neutralized or SDS-treated samples were fractionated under neutral pH and was reproducible when the 150-kDa complex was isolated, treated with acid or SDS, and rechromatographed. ALS in adult sera more tightly correlated with IGFBP-3 than IGF-I or IGF-II. The total levels (mean +/- SD) were 16.7 +/- 3.7 mg/L in 22 normal subjects, 28.3 +/- 8.1 mg/L in 20 acromegalic patients, and 9.5 +/- 3.8 in 32 GH-deficient adults. Little or no ALS was detectable in amniotic fluid, cerebrospinal fluid, seminal plasma, or milk, whereas high levels were present in synovial fluid. The development of ALS enzyme-linked immunosorbent assay should greatly facilitate further investigations of this unique glycoprotein.