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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.

Insulin-like growth factor-I (IGF-I) has mitogenic and anti-apoptotic effects on breast cancer cells. Epidemiologic studies have shown that high plasma levels of IGF-I and low levels of IGF binding protein (BP)-3 are associated with increased risk of breast cancer in premenopausal women. The actions of IGF-I are mediated through the IGF-I receptor (IGF-IR) and are regulated by IGFBPs. In circulation, most of the IGF-I binds to IGFBP-3, and binding of IGF-I to IGFBP-3 inhibits the actions of IGF-I. Since free IGF-I, which does not bind to IGFBPs, can readily cross the endothelial barrier to interact with IGF-IR, circulating free IGF-I is thought to be more relevant to the biologic activity of IGF-I. To examine the association of free IGF-I with breast cancer, we compared free IGF-I levels between 40 newly diagnosed breast cancer patients and 40 age- and race-matched healthy controls. Plasma levels of free IGF-I, total IGF-I and IGF-II, as well as total, intact and fragment IGFBP-3, were measured using commercial immunoassay kits. The association between IGF-I and breast cancer was examined using the conditional logistic regression analysis. Analysis of correlation (Spearman) showed that free IGF-I was correlated with total IGF-I and IGFBP-3 but not with IGF-II. The odds ratios for breast cancer patients having high plasma IGF-I (≥median) after adjusting for menopausal status and IGFBP-3 were 2.00 (p < = 0.376) for total IGF-I and 6.31 (p < = 0.047) for free IGF-I. A high ratio of IGF-I to IGFBP-3 was also associated with breast cancer (p < 0.05). No association was found for IGF-II, nor for total, intact and fragment IGFBP-3. The findings of this study suggest that measuring free IGF-I in circulation is more useful than measuring total IGF-I with respect to evaluation of an association between IGF-I and breast cancer risk. © 2001 Wiley-Liss, Inc.

Insulin‐like growth factors (IGFs) and IGF binding proteins (IGFBPs) play an important role in cell growth and differentiation. Clinical and epidemiological studies have indicated that measuring IGFs and IGFBPs in blood has potential implications in assessing growth‐related abnormalities and risks of certain types of cancer. To facilitate the application, we reported a large collection of reference ranges of IGFs and IGFBPs in normal population and evaluations of these molecules in serum and plasma as well as the impact of freeze‐thaw cycles on the measurement. IGF‐I, IGFBP‐3 and ALS showed a similar pattern of change associated with age. Levels of these molecules were low at birth and increased with age through puberty. After puberty the levels declined slowly with age. Overall, IGF‐I, IGFBP‐3 and ALS were slightly higher in females than in males. Free IGF‐I accounted for about 1% of the total IGF‐I and its variation with age was similar to total IGF‐I. IGF‐II levels were also increased with age from birth to puberty, but became stable after puberty. There was little difference in IGF‐II levels between genders. IGFBP‐2 levels declined with age from birth to puberty. Levels of IGFBP‐6 in contrast were increased with age. These IGF binding proteins were higher in males than in females. IGFs, IGFBP‐3 and ALS were 5‐10% higher in serum than in plasma. IGFBP‐2 and IGFBP‐6 differed substantially between serum and plasma. Freeze‐thaw treatment up to five cycles had little impact on plasma levels of IGFs and IGFBP‐3. Our observations suggest that levels of IGFs and their binding proteins are varied with age, gender, and types of specimen and that these variations need to be taken into consideration when IGFs and their binding proteins are utilized in clinic and research. J. Clin. Lab. Anal. 13:166–172, 1999. © 1999 Wiley‐Liss, Inc.

Insulin-like growth factors (IGFs) are potent mitogens involved in the regulation of cell proliferation and apoptosis. The action of IGFs is mediated through a specific cell membrane receptor (IGF-IR), and the interactions between IGFs and this receptor are regulated by IGF-binding proteins (IGFBPs). IGFBP-3 is one such protein which either suppresses or enhances the actions of IGFs. Findings from most in vitro studies suggest that IGFBP-3 inhibits breast cancer cell growth and facilitates apoptosis, but clinical studies have found that high levels of IGFBP-3 in breast cancer tissues are associated with unfavourable prognostic indicators of the disease, such as large tumour size, low levels of steroid hormone receptors, elevated S-phase fraction and DNA aneuploidy. To further examine the role of IGFBP-3 in breast cancer recurrence and survival, we conducted the following nested case-control study. From a cohort of 1,000 women treated surgically for primary breast cancer, we consecutively selected 100 patients who developed recurrent disease after surgery and 100 age- and year of diagnosis-matched patients who had no relapse. Concentrations of IGFBP-3 in breast tissue extracts were determined with an ELISA. Inverse correlations of IGFBP-3 were revealed with estrogen receptor expression and patient age but not with tumour size or S-phase fraction. Levels of IGFBP-3 in breast tissues were slightly higher in the recurrent patients than in controls, but the differences were not statistically significant. No significant association was found between IGFBP-3 and breast cancer recurrence. Survival analysis, however, indicated that the risk of death was increased with higher IGFBP-3 levels, and the association was independent of other prognostic markers. In conclusion, our results demonstrate that high levels of IGFBP-3 are associated with unfavourable prognostic features of breast cancer.

We developed a specific, simple, and rapid RIA for the direct quantification of estrone sulfate (E1S) and established its performance characteristics. The assay has a dynamic range of 0.05–90 μg/L with a detection limit of 0.009 μg/L. Intraassay CVs were 9.2%, 4.5%, and 4.6% at 0.35, 9.0, and 60 μg/L, respectively. Interassay CVs were 8.8%, 5.1%, and 5.5% at 0.076, 0.5, and 12 μg/L, respectively. Linearity of dilution studies showed values of 80–105% of expected, and recovery of E1S added to serum samples ranged from 82% to 102%. Cross-reactivities with structurally related estrogens were

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.

Insulin-like growth factor (IGF) I and IGF binding proteins (IGFBPs) modulate metabolic activity and tissue repair and are influenced by nutritional status. IGF-I circulates in free, ternary [IGF-I + IGFBP-3 + acid labile subunit (ALS)], and binary (IGF-I + IGFBP) molecular complexes, and the relative proportions regulate IGF-I extravascular shifting and bioavailability. This study examined the hypothesis that sustained physical activity and sleep deprivation superimposed on a short-term energy deficit would alter the IGFBP concentrations and alter the proportions of IGF-I circulating in ternary vs. binary molecular complexes. Components of the IGF-I system (total and free IGF-I; IGFBP-1, -3, and ALS; nonternary IGF-I and IGFBP-3), biomarkers of metabolic and nutritional status (transferrin, ferritin, prealbumin, glucose, free fatty acids, glycerol, beta-hydroxybutyrate), and body composition were measured in 12 men (22 +/- 3 yr, 87 +/- 8 kg, 183 +/- 7 cm, 20 +/- 5% body fat) on days 1, 3, and 4 during a control and experimental (Exp) period. During Exp, subjects performed prolonged work (energy expenditure of approximately 4500 kcal/day) with caloric (1600 kcal/day) and sleep (6.2 h total) restriction. IGF-I and IGFBP-3 were measured by immunoassay before and after immunoaffinity depletion of ALS-based complexes (i.e., ternary complex removal). Exp produced losses in body mass (-3.0%), lowered total IGF-I (-24%), free IGF-I (-42%), IGFBP-3 (-6%), nonternary IGF-I (-27%), and IGFBP-3 (-16%), and increased IGFBP-1 (256%). No Exp effects were observed for ALS. No changes were observed in the proportion of IGF-I circulating in free ( approximately 1.2%), ternary ( approximately 87.4%), or nonternary ( approximately 11.4%) molecular complexes. During Exp, glucose concentrations were lower on day 3, but days 1 and 4 were statistically similar. In conclusion, during a short-term energy deficit in young, healthy men, 1). IGF-I system components differentially respond (both in direction and magnitude) to a given metabolic perturbation and 2). the relative proportion of IGF-I sequestered in ternary vs. nonternary molecular complexes appears to be well maintained.

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.

Accurate measurement of insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is important for precise definition of its physiological roles and potential diagnostic values. Because altered phosphorylation results in altered IGFBP-1 immunoreactivity, current assays may significantly underestimate or fail to detect physiological changes in the IGFBP-1 concentrations. We developed three ELISAs (ELISA 1–3) using a common capture but three different detection antibodies. IGFBP-1 in serum, synovial fluid (SF), cerebrospinal fluid (CSF), and amniotic fluid (AF) were measured before and after treatment with alkaline phosphatase (ALP). Among the methods, only ELISA-1 was unaffected by IGFBP-1 phosphorylation and generated identical results before and after ALP treatment. The serum and SF values by ELISA-2 and -3 were lower by ∼4- to 10-fold, but increased after ALP treatment to within 66–98% of those by ELISA-1. The medians in AF, and to a lesser extent in CSF, by all methods were similar and did not change significantly after dephosphorylation. ELISA-1 showed excellent correlation with ELISA-2, ELISA-3, and a commercial IGFBP-1 IRMA only after ALP-treated samples were analyzed by the comparative methods. ELISA-1 is highly specific for IGFBP-1 and demonstrated acceptable analytical performance characteristics.